Acetylcholine, melatonin, and potassium depolarization stimulate release of luteinizing hormone-releasing hormone from rat hypothalamus in vitro ( ABSTRACT We have examined the release of radioimmunoassayable luteinizing hormone-releasing hormone (LH-RH) from fragments of rat medial basal hypothalamus. These fragments were cultured overnight in medium containing serum and then preincubated in groups of three for 10 min in medium resembling cerebrospinal fluid in its electrolyte constituents and containing bacitracin. This was followed by 30-min incubation periods during which some of the hypothalami were exposed to test substances. Potassium depolarization, effected by the addition of 56 mM potassium chloride to the incubation medium, caused a marked stimulation in LH-RH release, but only in the presence of calcium. Acetylcholine at 10 nM and the parasympathomimetic anticholinesterase agent neostigmine at 1 ,uM markedly stimulated LH-RH release. Hexamethonium, a nicotinic antagonist, at 1 IAM abolished the acetylcholine-induced increment in LH-RH