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Transneuronal tracing techniques were used to identify spinal and brainstem neurons involved in the control of perineal muscles in the male rat. Two penile muscles, the bulbospongiosus and ischiocavernosus muscles, were injected with Bartha's strain of pseudorabies virus. After survival periods of 2, 4, and 5 days, the rats were killed and viral labeled neurons identified by immunohistochemistry. After a 2 day survival period, only pudendal motoneurons were labeled. More spinal and brainstem neurons were labeled at longer survival times. Putative spinal interneurons were found from T13 to S1. Large numbers of neurons were found in the lateral horn of the T13-L2 and L6-S1 segments which contain sympathetic and parasympathetic preganglionic neurons, respectively. However, retrograde labeling experiments verified that very few of the viral neurons were preganglionic neurons. Other labeled neurons were found in the intermediate cord, especially around the central canal. Relatively few labeled neurons were seen in the dorsal or ventral horn. In the brainstem, consistent labeling was seen in the ventrolateral medulla, raphe pallidus, and magnus, the A5 and locus ceruleus noradrenergic cell groups. Barrington's nucleus in the pontine tegmentum, the periaqueductal gray, and the paraventricular nucleus of the hypothalamus. The transneuronal labeling was consistent with what is currently known of the central nervous system (CNS) control of the perineal muscles.