The present experiments were performed to determine the temporal pattern of expression of nitric oxide synthase (NOS) immunoreactivity in cells and fibers in the lumbosacral spinal cord during early postnatal development and to examine the relationship between NADPH-diaphorase (NADPH-d) activity and NOS-immunoreactivity (IR). At postnatal days 0-1 and 4-5, NADPH-d and NOS-IR were detected in L6-S1 segments of the spinal cord in cells and fibers in the region of the sacral parasympathetic nucleus (SPN), dorsal commissure and around the central canal but were absent in the superficial layers of the dorsal horn. Fiber staining on the lateral edge of the dorsal horn (the lateral collateral pathway, LCP) in a region containing primary afferent projections from the pelvic viscera and in a fiber tract in the dorsolateral funiculus was also not detectable. At days 4-5 some stained cells were detected in the deeper laminae of the dorsal horn. At postnatal days 10-12 and 20-22, cells in the region of the SPN, around the central canal and in the superficial laminae of the dorsal horn exhibited NADPH-d and NOS-IR. NADPH-d and NOS-IR fiber staining in the superficial laminae of the dorsal horn and the dorsolateral funiculus was observed at postnatal days 10-12 and increased in staining intensity by postnatal days 20-22. NADPH-d fiber staining in the LCP was not prominent at postnatal days 10-12; however, prominent fiber staining at this site did occur by postnatal days 20-22 and in adult animals. In postnatal days 20-22 and in adult animals NADPH-d activity and NOS-IR had a similar distribution except in the LCP where NADPH-d stained fibers did not exhibit NOS-IR. These data indicate that NADPH-d and NOS-IR in the spinal cord exhibit marked changes during the early postnatal development. The changes in afferent projections in the LCP may be related to maturation of visceral reflex pathways including micturition.