Abstract Administration of human chorionic gonadotropin (hCG) to hypophysectomized immature rats caused a rapid reduction in ovarian microsomal 17α-hydroxylase/C17,20-lyase activity (cytochrome P450 17α) with a concomitant large increase in serum progesterone (P 4) level. Pretreatment with cycloheximide (Cycio) or aminoglutethimide (Ag) prevented these effects of hCG, while Actinomycin d (Act-D) or Azastene, an inhibitor of 3-hydroxysteroid dehydrogenase, were ineffective. In ovaries with enzyme activity increased by 48 h exposure to pregnant mare's serum gonadotropin, hCG also caused a large decrease in enzyme activity but only after a lag period of about 2 h: P 4 levels were increased simultaneously. Administration of Cyclo. or puromycin (Puro) caused a loss of enzyme activity without changing P 4 levels, but both inhibitors prevented some of the loss of activity and rise in P 4 induced by hCG. AG and Act D completely inhibited the enzyme reducing action of hCG, as well as the increase in P 4 synthesis, in these animals. P4 applied directly onto one ovary of an animal given hCG plus AG reduced enzyme activity by 69%. The results are consistent with the interpretation that increased substrate concentration is one of but not the only important factor in loss of hydroxylase/lyase activity induced by a sudden large increase in luteinizing hormone activity.