Testicular steroidogenic enzymes in the microsomal fraction from immature pigs were investigated for the effects of phospholipids of known structure on androgen and 16-androstene biosynthesis. Untreated (control) microsomes metabolised pregnenolone to 17-hydroxypregnenolone, DHA and small quantities of progesterone, 17-hydroxyprogesterone, androstenedione and testosterone; and to 5,16-androstadien-3β-ol (andien-β) and 4,16-androstadienone (dienone) in the 16-androstene pathway. Phosphatidyl( P)-serine, P-glycerol, P-ethanolamine, P-inositol, P-choline and phosphatidic acid did not significantly alter the 17-hydroxylase/C-17,20 lyase or “andien-β-synthetase” activities. Thus, the C 21 side-chain cleavage reactions appeared not to be dependent upon phospholipids for optimal activity. The conversion of pregnenolone to 4-ene steroids (progesterone, 17-hydroxyprogesterone, androstenedione and testosterone) was inhibited by dilinoleoyl-phosphatidyl-choline, choline, but other phospholipids tested were without effect. On the other hand, the conversion of andien-β to dienone was inhibited by P-serine, P-inositol and P-cholines with short saturated or lung polyunsaturated acyl chains. Therefore, the presence of these phospholipids in pregnenolone incubations had different consequences for 3β-hydroxysterid dehydrogenase-isomerase activities. It is concluded that subtrate specific 3β-HSD-isomerases exist for androgen and 16-androstene biosynthesis and that phospholipids may play an intrinsic role in their catalytic activity.