Nitric oxide has been proposed to act as an intercellular messenger in central brainstem circuits controlling gastrointestinal motility. In particular, a subpopulation of preganglionic vagal neurons of the dorsal motor nucleus of the vagus have been shown to be reduced nicotinamide adenine dinucleotide phosphate(NADPH)-diaphorase positive; NADPH-diaphorase positive preganglionic fibers are also known to make contact with enteric neurons in the stomach. No studies, however, have correlated the neurochemical phenotype of preganglionic vagal neurons to their stomach target. The purpose of this study was to identify the subpopulation of nitric oxide synthase positive vagal neurons projecting to the stomach. Fluorescent retrograde tracers were injected in the fundus, corpus or antrum (Rhodamine beads) or painted on the anterior gastric branch of the vagus (DiI); five to 15 days later the brainstem was processed for nitric oxide synthase immunoreactivity. Of the 532 DiI-labeled neurons from the vagal anterior gastric branch, 25 (4.7%, n=5 rats) were co-localized with nitric oxide synthase immunoreactivity. Of the neurons labeled following injection of rhodamine beads in the antrum (N=231 neurons, n=5 rats) or corpus (N=166 neurons, n=4 rats) only three neurons showed nitric oxide synthase immunoreactivity (two in antrum and one in corpus, respectively). Conversely, 26 of 222 neurons (12%, n=7 rats) labeled following injection of rhodamine in the fundus showed nitric oxide synthase immunoreactivity. These results provide evidence for a discrete phenotypic subpopulation of vagal motoneurons that project to the gastric fundus, and suggest that these neurons may be the ones involved in the receptive relaxation reflex.