Male Sprague-Dawley rats, with their pelvic and hypogastric nerves transected, were infected with pseudorabies virus (PRV) injected into the external urethral sphincter. Animals were sacrificed at 2, 2.5, 3, and 4 days postinfection. Spinal cord and brain tissue were sectioned and processed by immunohistochemical techniques with antisera against PRV and choline acetyl transferase (CAT). At 2 days postinfection, virus-labeled neurons were found in the ventrolateral divisions of Onuf's nucleus and in the dorsal gray commissure (DGC). At progressively later incubation times, labeled neurons were found in the intermediolateral regions, the superficial layer of the dorsal horn, and the brainstem, in particular, the pontine micturition center. PRV/CAT-positive neurons were only found in Onuf's nucleus. Preganglionic neurons in the L6-S1 intermediolateral regions were CAT positive but PRV negative, thus suggesting that they are interneurons, not sacral parasympathetic preganglionic neurons. After 4 days, virus had spread to neurons in the paraventricular, preoptic, and even cortical regions. The distribution of these PRV-labeled brain neurons strongly resembled that obtained after the injection of PRV into the urinary bladder (Nadelhaft et al. [1992] Neurosci. Lett. 143:271-274). In both cases, neurons were labeled in the DGC in the spinal cord. The data therefore suggest that neurons in the DGC may be involved in the integrated control of the bladder and the external urethral sphincter.