Prostaglandin E2 (PGE2) stimulates steroidogenesis in vitro in human corpus luteum slices, to a similar extent as hCG. This prostaglandin, as well as hCG and hLH also stimulates the incorporation of [8-3H] adenine into [8-3H] cyclic AMP in this in vitro system and this stimulation appears to be specific and dose related for these substances. The increase of [8-3H] cyclic AMP accumulation was found to be due to the stimulation of the synthetic adenyl cyclase enzyme system rather than the inhibition of the destructive phosphodiesterase enzyme system. Throughout the study, corpora lutea of the menstrual cycle were shown to be much more responsive to stimulation (hCG, LH, PGE2) in terms of steroid synthesis and cyclic AMP formation than corpora lutea of pregnancy. The reason for this difference between the two types of corpora lutea is not clear at this time but does not appear to be due to a deficiency in the adenyl cyclase system of pregnancy corpora lutea. The data of the present study brings our understanding of the mechanism of action of gonadotropin on the human corpus luteum in vitro up to the level of our current knowledge for the bovine corpus luteum.