OBJECTIVE The central subnucleus of the nucleus of the solitary tract has been implicated in central reflex control of esophageal peristalsis. This study determined the presence of nitric oxide synthase in the brain stem circuit controlling esophageal peristalsis by combining transsynaptic retrograde tract tracing with pseudorabies virus and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH) histochemistry. METHODS Virus was injected into the esophagus of 10 of 15 rats. After 60-63 hours, brain sections were processed for viral immunofluorescence and NADPH histochemistry. RESULTS Fluorescent neuronal labeling was limited to the compact formation of the nucleus ambiguus and the central subnucleus of the nucleus of the solitary tract. Most fluorescence-labeled neurons in the central subnucleus stained positively for NADPH (double labeled). In the compact formation, there were almost no double-labeled neurons; however, NADPH-stained terminals surrounded fluorescence-labeled motoneurons. CONCLUSIONS NO synthase is present in premotor neurons of the central subnucleus of the nucleus of the solitary tract that innervate esophageal motoneurons in the compact formation of the nucleus ambiguus. NADPH staining in both somata and terminals of esophageal premotor neurons suggests that NO is involved in neurotransmission in the central subnucleus and at the site of synaptic contact between esophageal premotor neurons and motoneurons in the compact formation of the nucleus ambiguus.