The effect of acetylcholine on the neurointermediate lobe β-endorphin secretion was studied in the neonatal and in the adult rat in vitro. Acetylcholine stimulated β-endorphin secretion from the 2-day- and 5-day-old neurointermediate lobe, the effect was dose dependent and more pronounced in the presence of the cholinesterase inhibitor eserine. The 10-day-, the 21-day-old and the adult rat neurointermediate lobes did not respond to acetylcholine, even in the presence of eserine. Basal β-endorphin secretion was elevated by the D 2 receptor antagonist sulpiride, but acetylcholine was without effect in the 10-day-old and in the adult neurointermediate lobe even after dopamine receptor blockade. The β-endorphin stimulatory response to acetylcholine was diminished by the M 1 muscarinic receptor antagonist pirenzepine and blocked by the M 3 > M 1 antagonist 4-diamino-phenyl-piperidine (4-DAMP). The selective M 2 antagonist methoctramine and nicotine had no effect. These data indicate that the neurointermediate lobe β-endorphin secretion is under special muscarinic cholinergic regulation for a relatively short time after birth. The disappearance of this stimulatory cholinergic effect in later life might be due to changes in the intracellular secretory machinery in the IL and/or to the uncoupling of the cholinergic receptors from the intracellular signal transduction system(s) responsible for the stimulated secretion in the rat melanotrope cells.