Nowadays the incidence of various enzymatic pathologies, like lactase deficiency – inability to digest milk sugar tends to grow. According to WHO report, 10 to 80% of humans representing diverse population groups suffer from lactase deficit in the body. Lactose not cleaved in the small intestine is translocated into distal part of the tract resulting in increased osmotic pressure, retention of liquid, meteorism, swollen abdomen, and diarrhea. The clinical syndrome is caused by underproduction of β-galactosidase enzyme hydrolyzing lactose and different galactosides – oligo- and polysaccharides, glycopeptides, glycoproteins, mucopolysaccharides, splitting off β-D-galactose residue. Major method to control lactase deficiency is exclusion or limited consumption of milk and derived products coupled to diet enrichment with food additives containing commercial β-galactosidase. The studies have shown that several bacteria – constituents of normal gut microbiota, including genera Lactobacillus and Bifidobacterium are capable to synthesize β-galactosidase in amounts sufficient to promote assimilation of lactose by human organism. It was found that the majority of bifidobacterial cultures produce series of β-galactosidase isoenzymes performing similar functions and differing in physical-chemical properties. Each isoenzyme is responsible for specific hydrolytic or synthetic reaction related to lactose and various galactosides metabolism. Our predominant research interest is focused on inclusion of β-galactosidase-generating bifidobacteria into starter microbial formulas to facilitate complete lactose utilization during fabrication of fermented dairy product. Lactose hydrolysis raises its digestibility in gastrointestinal system, intensifies thrice sweet taste of the end product. Moreover, transglycosylating action of β-galactosidase on milk sugar releases such compounds as oligosaccharides upgrading nutritive value of the products and stimulating development of bifidoflora. Strain Bifidobacterium adolescentis resulting from induced mutagenesis with subsequent selection for β-galactosidase activity level served as object of our studies. It was established that the mutant strain produce extra- and intracellular β-galactosidases. The level of bacterial β-galactosidase synthesis on media of diverse composition was investigated. Maximum enzyme yield were recorded in B. adolescentis culture utilizing lactose as carbon source. The optimal conditions for hydrolytic activity of extracellular and intracellular enzymes were defined at pH 5.0 - 6.5 and temperature 50°С. The optimal for β-galactosidase transglycosylating action were observed at pH 7.0–7.5 and temperature range 37–50°C. Intracellular enzymes maintained activity at low temperatures (+4 to 8°C). Application of probiotic bacteria with β-galactosidase activity as ferment ingredients (ingredients of starter cultures) will contribute to solution of lactase deficiency problem and will add valuable properties and advantages to dairy products by improving flavor and technological merits thereof.