Immunoreactivities against anti-bombyxin and anti-muscarinic acetylcholine receptor on sequential sections of the pupal brain of Bombyx mori showed that bombyxin-producing neurosecretory cells expressed the muscarinic acetylcholine receptor. Carbachol at 100 mM induced isolated bombyxin-producing neurosecretory cells to depolarize in the range of 5 to 20 mV and the carbachol-induced depolarization was blocked with 1 mM atropine (muscarinic acetylcholine antagonist). These results suggested that a direct transmission to regulate release of bombyxin occurs on the bombyxin neurosecretory cells. In fact, the release of bombyxin from the brain-corpus cardiacum-corpus allatum complex was induced with carbachol and inhibited with atropine, indicating that the release of bombyxin is, at least partially, regulated by muscarinic transmission.