The neural control of renal function is exerted by the central nervous system via sympathetic innervation of the kidneys. To determine the extent to which the control of the two kidneys is provided by the same brain neurons, the central circuitry involved in the innervation of both kidneys was characterized in individual rats by dual viral transneuronal tracing using isogenic recombinant strains (PRV-152 and BaBlu) of pseudorabies virus. Prior to dual tracing, the neuroinvasive properties of PRV-152 and BaBlu were characterized by conducting parametric studies, using the two kidneys as an anatomical model, and comparing the pattern of infection with that obtained following injection of the parental strain, PRV-Bartha, into the left kidney. Once the optimal concentrations of virus required to obtain equivalent infection were established, PRV-152 and BaBlu were injected into the left and right kidney, respectively, in the same rats. Immunocytochemical localization of viral reporter proteins at different postinoculation times allowed us to determine the sequence of infection in the brain, as well as to quantify dual- and single-labeled neurons in each infected area. Neurons that influence autonomic outflow to one or both kidneys coexist in all brain areas involved in the control of the sympathetic outflow to the kidneys at every hierarchical level of the circuit. The proportions of dual-infected neurons with respect to the number of total infected neurons varied across regions, but they were maintained at different survival times. The pattern of infection suggests that the activity of each kidney is controlled independently by organ-specific neurons, whereas the functional coordination of the two kidneys results from neurons that collaterize to modulate the sympathetic outflow to both organs. The advantages of using an anatomical symmetrical system, such as the two kidneys, as an experimental approach to characterize PRV recombinants in general are also discussed. J. Comp. Neurol. 471:462–481, 2004. © 2004 Wiley-Liss, Inc.