Immunoreactivity to nitric oxide synthase (NOS-IR) and choline acetyltransferase (ChAT-IR) was detected in the adult rat spinal cord using the avidin-biotin-peroxidase technique. Intensely stained NOS-positive neurons with cell processes were observed in the intermediolateral cell column of the thoracic and sacral segments and around the central canal of all segments. These areas also contained ChAT-IR neurons. A number of small- to medium-sized NOS-IR cells were noted in the superficial and deeper laminae throughout the entire cord. NOS-IR was not detected in the ventral horn motoneurons, which were, however, ChAT-IR. The results indicate that NOS-IR is present in autonomic preganglionic neurons and in selected neurons in the dorsal horn and lamina X, but appears to be absent in motoneurons.