Cardiovascular diseases and ways of their therapy are still one of the central issues of biomedical research. Such diseases arise due to irregularities in protease cascade system which controls the density of human blood known as hemostasis system. Treatment in these cases involves replacement therapy, however the enzymes used for treatment are very expensive or not specific enough [1], therefore the search for new sources of such proteases is still relevant. Extracellular proteases of micromycetes, specifically activating the enzymes of the hemostatic system are considered a promising substitution [2]. It was shown that proteases of enthomoand nematopathogenic fungi might be a prospective source of thrombolytic proteases [3]. During our previous research we have done a screening among such fungi and a strain of Purpureocillum lilacinum k1 was selected as a promising one. This strain secreted a protease with ability to cleave a specific chromogenic peptide substrate of human activated protein C (human APC-like activity) [4]. This study was focused on the selection of optimal cultivation conditions and checking an influence of various nitrogen sources and their concentrations on production of enzyme with APC-like activity. During the experiment culture was grown on the medium with a must and peptone for 48 h and then 3% of biomass was transferred to media with variations of nitrogen sources in composition. Then an activity with chromogenic peptide substrates (CPS) of activated protein C (pyroGly-Pro-Arg-pNA), tissue plasminogen activator (H-D-Ile-Pro- Arg-pNA) and thrombin (Tos-Gly-Pro-Arg-pNA) was measured on 13th day. The most significant APC-like activity was registered in medium with 1% of fish flour hydrolysate. Quite high rate was shown on a medium with a combination of sodium nitrate (0.2%) and ammonium sulfate (0.2%), despite both those forms of nitrogen decreased a synthesis of enzyme being separately. Nevertheless, a media with hydrolyzed fish flour seems to be the most suitable due to its simple and cheap composition. Moreover, explored side activities with CPS of thrombin and tissue plasminogen activator decreased with increasing concentration of fish hydrolyzate. This distinguishes fish flour hydrolysate as a factor that allows achieving a slightly higher selectivity to obtain investigated enzyme.