Despite considerable interest in the neural mechanisms that regulate muscle blood flow, the descending pathways that control sympathetic outflow to skeletal muscles are not adequately understood. The present study mapped these pathways through the transneuronal transport of two recombinant strains of pseudorabies virus (PRV) injected into the gastrocnemius muscles in the left and right hindlimbs of rats: PRV-152 and PRV-Bablu. To prevent PRV from being transmitted to the brainstem via motor circuitry, a spinal transection was performed just below the L2 level. Infected neurons were observed bilaterally in all the areas of the brain that have previously been shown to contribute to regulating sympathetic outflow: the medullary raphe nuclei, rostral ventrolateral medulla (RVLM), rostral ventromedial medulla, A5 region, locus coeruleus (LC), nucleus subcoeruleus (SC), and the paraventricular nucleus of the hypothalamus (PVN). The RVLM, the brainstem region typically considered to play the largest role in regulating muscle blood flow, contained neurons infected following the shortest post-inoculation survival times. Approximately half of the infected RVLM neurons were immunopositive for tyrosine hydroxylase, indicating that they were catecholaminergic. Many (47%) of the RVLM neurons were dually-infected by the recombinants of PRV injected into the left and right hindlimb, suggesting that the central nervous system has a limited capacity to independently regulate blood flow to left and right hindlimb muscles.