Abstract We describe the distribution and characteristics of nitric oxide synthase-containing neurons in rat spinal cord using a polyclonal affinity-purified antibody against rat cerebellar nitric oxide synthase. Numerous neurons were stained throughout the entire rostrocaudal extent of the spinal cord. Cell bodies, dendrites and axons stained in a uniform manner. Nitric oxide synthase immunoreactivity was intense in neurons of laminae I–IV and X throughout the entire spinal cord. Neurons in the intermediolateral cell column of the thoracic and lumbar spinal cord were also intensely stained for nitric oxide synthase. The sacral cord demonstrated substantial nitric oxide synthase immunostaining within lamina VII. For the entire cord, scattered neurons in laminae V, VI, VII, and VIII were weakly positive. In addition, punctate nitric oxide synthase staining throughout laminae I, III and surrounding some large motor neurons in the ventral horn suggested the presence of nitric oxide synthase at synapses. Axons and dendritic terminals located in the gray and white matter were also stained. The majority of nitric oxide synthase positive neurons in the intermediolateral cell column were double-labelled by subcutaneously injected FluoroGold confirming that these cells were preganglionic autonomic neurons. Most NADPH-diaphorase-stained neurons were also nitric oxide synthase-positive. The distribution of nitric oxide synthase-containing neurons in spinal cord suggests that nitric oxide plays a role in spinal cord neurotransmission including: preganglionic sympathetic and parasympathetic, somatosensory, visceral sensory and possibly motor pathways. In particular, the autonomic nervous system appears enriched with nitric oxide synthase immunoreactivity. The precise role of each neuron type remains to be demonstrated in physiologic and pathophysiologic paradigms.