We employed retrograde axonal tracing techniques and nitric oxide synthase (NOS) and choline acetyltransferase (ChAT) immunohistochemistry to identify NOS-containing neuronal populations within the lumbosacral spinal cord and determine whether these project to the major pelvic ganglion in the adult male Sprague-Dawley rat. Immunohistochemical localizations of NOS included neurons situated at the L5 to S2 segments of the spinal cord, which corresponded to the sacral parasympathetic nucleus. Another prominent locus for NOS was a group of neurons identified in the L1 segment corresponding to the dorsal commissural nucleus. These regions correlated directly with preganglionic parasympathetic and sympathetic neuronal origins, respectively, which were established with ChAT colocalizations. Retrograde tracing verified the projection of these neurons to the pelvis. Additional neuronal localizations of NOS were observed throughout the intermediolateral cell column, involving the superficial laminae of the dorsal horn, in the region surrounding the central canal and occasionally in the medial area of the ventral horn. These results indicate that the regulation of pelvic visceral activity may involve NO-based neuronal mechanisms operating at the level of the lumbosarcal spinal cord.