Histochemical staining was used to demonstrate that intramural neurons of the gallbladder contain NADPH-diaphorase, and therefore are likely to produce nitric oxide. A subset of the neurons in the gallbladders of the guinea pig, gerbil, opossum, dog, and human stained positively for the enzyme. In the guinea pig, all neurons that were immunoreactive for vasoactive intestinal peptide (VIP), also contained NADPH-diaphorase. Furthermore, neurons that were immunoreactive for neuropeptide Y, which have been shown to be immunoreactive for substance P and somatostatin as well, rarely contained NADPH-diaphorase. It is suggested that the VIP/NADPH-diaphorase-containing neurons represent intrinsic inhibitory motor neurons of the gallbladder, and that these neurons may have a role in the relaxation of the muscularis during gallbladder filling.