For clarification of the effects of steroid concentration on steroidogenesis of adrenal microsomes, the kinetic parameters, K(m) and k(cat), were determined in the steady-state for progesterone and 17-alpha-hydroxyprogesterone metabolism catalyzed by P-450c21 and P-450(17-alpha,lyase) in guinea pig adrenal microsomes. At a high concentration of progesterone, it was equally metabolized by P-450C21 and P-450(17-alpha,lyase,) while at a low concentration, it was hydroxylated at 17-alpha-position with twice higher rate than at 21-position. 17-alpha-Hydroxyprogesterone is apparently metabolized preferentially by P-450C21 at any concentration. Although the productions of deoxycortisol and androstenedione from 17-alpha-hydroxyprogesterone were strongly inhibited by progesterone, androstenedione formation from progesterone was not inhibited by a high concentration of progesterone. The addition of liposomal P-450C21 to the reaction medium containing adrenal microsomes caused a decrease in the concentration of 17-alpha-hydroxyprogesterone released into the medium in the steady state reaction, but this had no effect on the activity of androstenedione formation from high concentrations of progesterone. It thus follows that androstenedione is produced by successive monooxygenase reactions without the release of 17-alpha-hydroxyprogesterone from P-450(17-alpha,lyase) at a high concentration of progesterone, which is the condition of the adrenal microsomes in vivo.