Steroidogenesis in the ovary and the effects of human chorionic gonadotropin (hCG) and prostaglandins (PGs) were examined. Ovaries from proestrous rats were incubated with 14C-acetate and the labeled steroids were extracted and separated by thin layer chromatography. On addition of hCG, incorporation of label into progesterone, 20 alpha-dihydroprogesterone and 17 alpha-hydroxyprogesterone was remarkably increased. PGF2 alpha and PGE2 addition also increased these steroids. Pregnenolone, androstenedione, testosterone and estradiol were not affected by hCG or either PG. Estrone was increased only by PGF2 alpha. When PGF2 alpha or PGE2 were used at the same time as hCG, the increase of progesterone, 20 alpha-dihydroprogesterone and 17 alpha-hydroxyprogesterone induced by hCG was reduced by both PGs in a dose dependent manner. These results show that hCG and PGs have stimulatory effects on steroidogenesis in proestrous rat ovary, and that PGs can inhibit this stimulatory effect of hCG.