The role of cAMP in the regulation of the amount and synthesis of cytochrome P-450 cholesterol sidechain cleavage (P-450_scc) and cytochrome P-450 17α-hydroxylase/C_17-20 lyase (P-450_17α) was investigated in mouse Leydig cell cultures. In the absence of cAMP, the amount of immunoreactive P-450_17α decreased to less than 5% by day 4 and was undetectable between days 7 and 11. In contrast, the amount of immunoreactive P-450_scc remained relatively constant throughout the same period. Treatment of Leydig cell cultures for 4 days with 0.05 mm 8-bromo-cAMP initiated on day 7 increased the amount of P-450_17α with relatively little effect on the amount of P-450_scc. The rate of de novo synthesis of each of the P-450 enzymes was studied by determining [³⁵S]methionine incorporation into newly synthesized protein. In the absence of cAMP, de novo synthesis of P-450_17α ceased while the rate of de novo synthesis of P-450_scc increased with time in culture between days 2 and 11. Treatment with cAMP initiated on day 7 of culture caused a timedependent increase in the rate of de novo synthesis of P-450_17α on days 9 and 11 equivalent to 40% and 60%, respectively, of that observed in freshly isolated Leydig cells. The rate of de novo synthesis of P-450_scc was increased 2-fold relative to untreated cultures on days 9 and 11. De novo synthesis of P-450_17α ceased when cAMP was removed on day 11 and restored when cAMP was added again on day 13 of culture. This treatment has little effect on de novo synthesis of P-450_scc. The capacity of Leydig cells for steroid production was determined by incubating cultures for 2 h with 1 mm 8-bromo-cAMP and measuring the amount of pregnenolone, progesterone, androstenedione, and testosterone produced. Total steroid production declined somewhat on day 2 and remained relatively unchanged during the remaining 9 days of culture in the presence or absence of chronic cAMP treatment. However, the pattern of steroid production changed markedly. In the absence of cAMP treatment, testosterone production decreased while progesterone production increased. Treatment of cultures with cAMP reversed this pattern of steroid production. These studies demonstrate that cAMP is essential for the synthesis of P-450_17α. P-450_scc, in contrast, is constitutively synthesized. Decreases in acute cAMPstimulated testosterone production in Leydig cell cultures, maintained in the absence of cAMP treatment, are not due to a reduced capacity for steroid production but reflect decreases in the amount of P-450_17α.