A very effective procedure was developed for separation of sex hormones of the delta 4 pathway using the microcolumn technique of high performance liquid chromatography (HPLC). A five-step gradient containing methanol, acetonitrile and water was used as a mobile phase, while Lychrosorb RP-18 with particles of 5 microns was used as a sorbent. Metabolism of steroid hormones and activity of 3 beta-steroid dehydrogenase, 17 alpha-hydroxylase, C17-20-lyase and 17-ketosteroid reductase were studied in Leydig cells of two mouse strains PT and A/He. The higher rate of steroid metabolism and of the enzymatic activity was detected in the mouse cells of PT strain as compared with those of A/He strain.