The possible participation of cholinergic mechanisms in the control of LH and LH-RH secretion has been studied using an in vitro procedure. Halved anterior pituitaries (AP) of normal adult male rats were incubated for 6 h either alone or in the presence of hypothalamic fragments (HF). At the end of the incubation period, LH was evaluated in the media using a biological assay. AP halves, when incubated alone, released small amounts of LH. The addition of HF to the media containing AP tissue did not change LH release. Acetylcholine (Ach) added to the incubation flasks containing only AP tissue did not increase LH output over the output of AP incubated without Ach. On the contrary, Ach significantly enhanced LH release when added to the incubation media of flasks simultaneously containing AP tissue and HF. Atropine had no effect on the release of LH from AP incubated alone. However the addition of atropine to the incubation media containing the halved AP, HF and Ach totally prevented the effects of Ach on LH release. Prostigmine enhanced the release of LH in the media containing AP plus HF. The LH-releasing effect of Ach in the AP plus HF coincubates was not influenced by the α-adrenergic blocking agent, phentolamine. These data indicate: (1) that from HFs incubated in vitro, Ach is able to release a factor (most probably LH-RH) which increases the secretion of LH from AP tissue; (2) that this effect of Ach follows the general rules of cholinergic systems (blockade by atropine, potentiation by prostigmine, etc.); and (3) that the LH-RH releasing activity of Ach is not linked to the liberation of catecholamines (dopamine, norepinephrine, etc.). These results suggest that cholinergic mechanisms play a role in the control of LH-RH release