The distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) was examined in the upper thoracic segment of the spinal cord in rat. Under the light microscope, NADPH-d positive cell bodies and fibers were readily detected in the following areas: 1) the dorsal horn; 2) the dorsolateral funiculus and lateral spinal neurons; 3) spinal autonomic region, consisting of the nucl. intermediolaterialis pars funicularis, nucl. intermediolateralis pars principalis, nucl. intercalatus spinalis and nucl. intercalatus pars paraependymalis; and 4) in the white matter lateral to the nucl. intermediolateralis pars funicularis. In the nucl. intermediolateralis pars principalis, the positive dendrites, running in bundles, were directed medially in the gray matter towards the central canal as well as laterally in the white matter towards the pia mater. The medially-directed positive dendrites fomed a subependymal plexus around the central canal. A dense bundle of NADPH-d positive fibers were also observed running longitudinally. Combined retrograde tracing with fluorogold and NADPH-d histochemistry study revealed that some of the NADPH-d positive neurons, due to their fluorescence labelling, were sympathetic preganglionic neurons that innervated the superior cervical ganglion. Under the electron microscope, the reaction products in the neurons of the nucl. intermediolateralis pars principalis were deposited in their nuclear envelope, rough endoplasmic reticulum, mitochondria and Golgi apparatus. In the neuropil, three types of synaptic configurations were observed: between NADPH-d negative axon terminals and NADPH-d positive dendrites, between NADPH-d positive axon terminals and NADPH-d negative dendrites, and between NADPH-d positive axons terminals and NADPH-d positive dendrites. These synaptic configurations suggest that the neurons are regulated by nitric oxide released from both pre- and post-synaptic elements. The sources of the NADPH-d positive axon terminals associated with the neurons remain unclear although the possibility of their being derived from supraspinal origins has to be considered. The ultrastructural demonstration of NADPH-d reaction product in the three major types of glial cells suggests that nitric oxide might be produced by these cells, but its functional significance awaits further investigation.