The distribution of nitric oxide synthase immunoreactivity (NOS-IR) and the changes in this distribution after peripheral axotomy were examined in lumbosacral afferent and preganglionic neurons (PGNs) innervating the pelvic viscera of the male rat. The visceral neurons in L6-S1 and L1-L2 dorsal root ganglia (DRG) and in the spinal cord were identified by retrograde axonal transport following injection of Fluorogold (FG) into the major pelvic ganglion (MPG). Axotomy was performed by removing the MPG on one side 2–4 weeks prior to sacrificing the animals. A differential distribution of NOS-IR was detected in DRG cells at different segmental levels of control animals. Significantly greater numbers of NOS-IR cells were present in thoracic (T8, T10, T12; 30–44 cell profiles/section) and rostral lumbar DRGs (L1-L2; 3–15 NOS-IR cell profiles/section) compared to caudal lumbosacral (L5-S1) DRGs (0.2–0.7 cell profiles/section). A significant increase in the number of NOS-IR cells was detected in the L6-S1 DRG (p < or = 0.001; 11 NOS-IR cell profiles/section) but not in the L2 or L5 DRG ipsilateral to axotomy. In these ganglia, an average of 37.0 +/- 4.0% (L6) and 20.6 +/- 2.2% (S1), respectively, of FG-labeled pelvic afferent neurons were NOS-IR compared to 1.1 +/- 0.5% (L6) and 2.5 +/- 1.4% (S1) contralateral to the axotomy. Following axotomy, a significantly greater percentage of dye-labeled pelvic visceral afferents in the L1 and L2 DRG also exhibited NOS-IR in comparison to the contralateral side. Following axotomy, NOS-IR fibers were detected along the lateral edge of the dorsal horn extending from Lissauer's tract to the region of the sacral parasympathetic nucleus (SPN) on the ipsilateral side of the L6 and S1 spinal segments. These NOS-IR fibers were not detected in adjacent spinal segments (L4, L5, or S2). Axotomy also changed the numbers of NADPH-d-positive and NOS-IR cells in the region of the SPN in the L6 spinal segment. Contralateral to the axotomy 38.3 +/- 4.0% of PGNs in the L6 spinal segment were colabeled with NOS-IR; however, ipsilateral to axotomy, a significantly greater percentage (61.0 +/- 3.0%; p < or = 0.01) of PGNs exhibited NOS-IR. Axotomy did not alter the distribution of PGNs in the S1 segment exhibiting NOS-IR. These results indicate that NOS-IR in visceral afferent and PGNs is plastic and can be upregulated by peripheral nerve injury.