Bovine corpora lutea slices were incubated with prostaglandins and gonadotropins. All the prostaglandins tested (PGE2, PGE1, PGF2α and PGA1) were found to be steroidogenic, stimulating both the production of progesterone as measured in micrograms and the incorporation of radioactivity from acetate-1-14C. Prostaglandin E2 (PGE2) gave the greatest effect, being approximately half as effective as luteinizing hormone (LH) on a molar basis. There were similarities between gonadotropin and prostaglandin. Cycloheximide (1 mm) equally blocked the steroidogenic response to both PGE2 and LH, the specific activities of the progesterone formed in the presence of prostaglandins and that formed in the presence of gonadotropins were approximately of the same order of magnitude, the time-response curves of PGE2 and LH were similar, and there was no additive effect when prostaglandins were added to luteal slices incubated with saturating doses of either LH or human chorionic gonadotropin (HCG). These results are in contrast to the in vivo luteolytic effect of PGF2α, and it is unlikely, therefore, that the luteolytic effect of PGF2α is due to a direct inhibition of luteal steroidogenesis. Thus, the in vitro and in vivo effects of prostaglandins may represent 2 different mechanisms of action.