Preganglionic neurons in the spinal cord of the rat were labelled retrogradely with Fluoro-gold and the spinal cord stained for NADPH diaphorase. The majority of both sympathetic and sacral parasympathetic preganglionic neurons showed staining for NADPH diaphorase. NADPH diaphorase-positive neurons were located more laterally in the intermediate zone than were preganglionic neurons lacking NADPH diaphorase staining. The recent evidence that identifies NADPH diaphorase as nitric oxide synthase raises the possibility that some spinal preganglionic neurons may synthesize nitric oxide.