Treatment of aggregate cell cultures of 14-day-old rat pituitary for 40 h with purified human (h) POMC(1-76) dose-dependently augmented the number of DNA replicating lactotrophs as estimated by autoradiography of [3H]-thymidine (3H-T) incorporation in cells immunostained for prolactin (PRL). No such effect was seen on the total number of 3H-T labelled cells (the majority of which did not contain any pituitary hormone in a detectable amount) or on the total number of lactotrophs. The effect of hPOMC(1-76) on 3H-T incorporation in lactotrophs was blocked by concomitant treatment with anti-hPOMC(1-76) monoclonal and polyclonal antibodies cross-reactive with rat POMC(1-74). The latter anti-hPOMC(1-76) antibodies also decreased the number of 3H-T incorporating lactotrophs in the absence of hPOMC(1-76). Gamma3-MSH, which is the C-terminal domain of hPOMC(1-76), mimicked the effect of hPOMC(1-76) on 3H-T incorporation in lactotrophs but its potency was lower than that of hPOMC(1-76). Other melanocortin (MC) peptides such as alpha- and beta-MSH were also effective but were less potent than gamma3-MSH. The difference in potency was not due to partial degradation of the peptides. hPOMC(1-76) did not affect 3H-T incorporation in other pituitary cell types. In contrast gamma3-MSH also augmented the number of 3H-T labelled somatotrophs and thyrotrophs. In the embryonic kidney 293 cell line stably transfected with the MC-3 receptor, gamma3-MSH (10 nM) augmented cAMP formation up to 30 times. In contrast, hPOMC(1-76) (100 nM) was inactive in this test system, indicating this peptide is not an agonist at the MC-3 receptor. The present investigation further supports the role of rat POMC(1-74) as a paracrine growth factor in the development of lactotrophs. The active core of POMC(1-76) does not seem to be restricted to its C-terminal domain gamma3-MSH as the latter peptide displays a growth promoting effect that is different from that of POMC(1-76): it is less potent, it is not specific for lactotrophs and whereas the effect of gamma3-MSH may be mediated by the MC-3 receptor that of POMC(1-76) is not.