The genus Physalis (fam. Solanaceae) is known as a source of withasteroids [1], mainly withanolides and physalins. Many of these compounds show anti-inflammatory, antitumor, hepatoprotective, immunomodulatory and cytotoxic activities [1, 2]. In our studies we elucidated the chemical structures of steroidal glycosides cholestane series from the seeds Physalis floridana Rydb. By gel filtration on Sephadex and adsorption-partition chromatography on silica gel, from the sum of glycosides obtained from the methanol extract, individual glycosides, named by us floridanoside B (1) and C (2) were isolated Each of floridanosides was subjected to complete acid hydrolysis, which resulted in the isolated aglycone with b.p. 198 – 199°C, [α]20 D 89.7°, (c 0.4, CHCl3), IR spectra: λmax: 3450, 2970, 1635, 1255, 1160, 1030 cm–1. The ESI-MS mass spectrum showed a major ion peak at m/z 435 [M + H]+. Therefore, its molecular formula is represented by C27H46O4, which was confirmed by HR-MALDI-MS. In the result of acid hydrolysis in the oligosaccharide part of each glycoside by TLC in the presence of authentic samples glucose and rhamnose were identified. The 1H NMR spectrum of aglycone showed signals for three methyl groups at δ 0.94 ppm (3H, s, Me-18), 0.8 ppm (3H, d, J=7.1 Hz, Me-21) and 1.28 ppm (3H, d, J=6.0 Hz, Me-27), two methine proton signals at δ 3.67 ppm (1H, m, H-3) and 3.65 ppm (1H, m, H-22) indicative of secondary alcoholic functions and one double bond at δ 5.68 ppm (1H, H-6), four methylene proton signals at δ ppm 3.58 (1H, m, H-19α), 3.88 ppm (1H, m, H-19β), 3.42 ppm (1H, m, H-26α) and 3.79 (1H, m, H-26β), ascribable to a primary alcoholic function. The C-25 configuration was deduced to be R based on the difference of chemical shifts (Δab=δaδb) of the geminal protons H2-26 (Δab=0.08 ppm). 13C NMR spectrum of aglycone displayed signals for 27 carbon atoms, which are clearly intended signals for three methyl groups at δ (ppm) 16.4 (C-18), 12.24 (C-21) and 17.7 (C-27), four oxygen bearing methine carbons at δ (ppm) 74.3 (C-22), 63.0 (C-19), 76.2 (C-3) and 70.6 (C-26), three quaternary carbons at δ (ppm) 43.2 (C-10), 41.7 (C-12) and 137.9 (C-5), seven methine carbons at δ (ppm) 34.4 (C-8), 36.9 (C-25), 41.5 (C-20), 52.1 (C-9), 53.8 (C-17), 22.9 (C-11) and 126.8 (C-6), and ten methylene carbons at δ (ppm) 24.9 (C-15), 29.9 (C-16), 30.2 (C-2), 30.9 (C-24), 39.8 (C-4), 33.1 (C-7), 33.0 (C-23), 34.7 (C-1), 43.8 (C-13) and 59.0 (C-14). Spectral data of 1H and 13C NMR analysis define the cholestane skeleton of the aglycone and on the basis of HSQC and HMBC correlations, it referred to the steroid compounds of the cholestane series with the following structure: (25R)- cholest-5-en-3β,19β,22β,26-tetraol. This genin, named by us floridagenin, is a new previously undescribed in the literature compound. According to NMR spectroscopy floridanoside B is a bioside, and floridanoside C  trioside. Indeed, in the 1H NMR spectrum in the range 4.8 – 6.5 ppm there are signals of two anomeric protons for 1 and three anomeric protons for 2 (the characteristic doublet splitting). Signals with chemical shifts δ (ppm) 5.26 for 1 and 5.26 and 4.87 for 2 based on the small value of constant of the spin-spin interaction (CSSI) (J1,2=1.5–1.6 Hz) refer to the anomeric proton of one rhamnose unit for 1 and two rhamnose residues for 2 and define their α-configuration. Anomeric proton signals with chemical shifts 3.27–4.56 ppm and a high value CSSI belong to glucose residues and define β-configuration of its glycoside bond. Types of glycosidе bonds in the carbohydrate part of each glycoside were established by a two-dimensional experiment ROESY (nuclear Overhauser effect in the rotating coordinate system). Thus, in the ROESY spectrum of 1 and 2, there are key cross-peaks at δ (ppm) 4.56 and 3.57 proving the spatial convergence of protons H-1 of glucose and H-3 of aglycone and uniquely determine the monosaccharide linked to the aglycone, at δ (ppm) 5.26 and 3.43, proving the linkage of rhamnose with C-2 OH group of glucose residues in 1, and in 2  at δ (ppm) 4.87 and 3.55, confirming the 1→4 bond between other residue of rhamnose and glucose, linked to genin. Based on these data floridanoside B is 3-O-α-L-ramnopyranosyl(1→2)-β-D-glucopyranoside-(25R)-cholest-5- en,3β,19β,22β,26-tetraol, and floridanoside C  3-O-{[α-L-ramnopyranosyl(1→4)]-[α-L-ramnopyranosyl(1→2)]-β- D-glucopyranoside}-(25R)-cholest-5-en,3β,19β,22β,26-tetraol. Both glycosides are new previously unspecified in the literature compounds.