The production of glucocorticoids and mineralocorticoids in the endoplasmic reticulum (ER) of the mammalian adrenal cortex are, to a great extent, regulated by the relative activities of the steroid 21-hydroxylase (P450c21) and steroid 17 alpha-hydroxylase (P450c17) enzymes. Progesterone can be 17 alpha-hydroxylated to yield 17 alpha-hydroxyprogesterone which, under certain conditions and in certain species, can be further lyased to adrostenedione by the same enzyme. P450c21 can 21-hydroxylate 17 alpha-hydroxyprogesterone to yield cortisol but also converts progesterone to corticosterone. Cytochrome b5 (cyt b5) can also participate in the regulation of adrenal microsomal steroid hydroxylase activities by changing the rates of the P450c21 and P450c17 reactions or by affecting the 17 alpha-hydroxylation:17,20-lyase ratio of progesterone, by P450c17. We investigated the metabolism of progesterone by sheep adrenal microsomes to identify the products of the different steroid hydroxylase activities in the ER and to investigate the influence of cyt b5 on progesterone metabolism using purified ovine cyt b5 and anti-cyt b5. The P450c17-activity in sheep adrenal microsomes is inhibited by the addition of purified cyt b5 while anti-cyt b5 IgG stimulates the 17 alpha-hydroxylation of progesterone. No 17,21-lyase-activity towards progesterone could be detected in sheep adrenal microsomes.