GnRH neurons form a final common pathway for the central regulation of reproduction. Although the involvement of acetylcholine in GnRH secretion has been reported, direct effects of acetylcholine and expression profiles of acetylcholine receptors (AChRs) still remain to be studied. Using immortalized GnRH neurons (GT1-7 cells), we analyzed molecular expression and functionality of AChRs. Expression of the mRNAs were identified in the order alpha 7 > beta 2 = beta 1 ae alpha 4 ae alpha 5 = beta 4 = delta > alpha 3 for nicotinic acetylcholine receptor (nAChR) subunits and m4 > m2 for muscarinic acetylcholine receptor (mAChR) subtypes. Furthermore, this study revealed that alpha 7 nAChRs contributed to Ca2+ influx and GnRH release and that m2 and m4 mAChRs inhibited forskolin-induced cAMP production and isobutylmethylxanthine-induced GnRH secretion. These findings demonstrate the molecular profiles of AChRs, which directly contribute to GnRH secretion in GT1-7 cells, and provide one possible regulatory action of acetylcholine in GnRH neurons.