The effects of 21-hydroxypregnenolone and related steroids such as deoxycorticosterone (DOC; 21-hydroxyprogesterone), cortisol, and corticosterone on progesterone 17α-hydroxylase activity by steroidogenic cytochrome P450 c17 (CYP17) were investigated. 21-Hydroxypregnenolone contains a hydroxyl group at C3 in the A cyclic hydrocarbon ring and a double bond at C5 in the B cyclic hydrocarbon ring, whereas DOC, cortisol, and corticosterone contain a ketone group at C3 and a double bond at C4 in the A cyclic hydrocarbon ring. No marked inhibition was observed for DOC, cortisol, and corticosterone at 100 μM concentration. Nonetheless, 21-hydroxypregnenolone exhibited competitive inhibition of progesterone 17α-hydroxylation activity by CYP17 with a Ki value of 36.4 µM. These results suggest that a hydroxyl group at C3 in the A ring and a double bond at C5 in the B ring in steroid hormones are important for the substrate recognition of CYP17.