The present report presents a series of experiments using Bartha's K strain of pseudorabies virus (PRV) designed to test the specificity of this virus as a retrograde transneuronal marker in the sympathetic nervous system of rats. Three experiments were performed. First, an injection of PRV was made in the anterior chamber of the eye, followed 24 hr later by an injection of WGA-HRP. PRV infected neurons in the superior cervical ganglion (SCG) always contained WGA-HRP. This dual labeling of SCG neurons suggests that the PRV cell body labeling does not nonspecifically infect nearby neurons. Second, after PRV injections into the pinna or eye, a specific segmental distribution of the transneuronal cell body labeling occurred in the intermediolateral cell column and related spinal areas. After eye injections, the majority of transneuronally labeled neurons were in the T1-T3 segments. After pinna injections, the majority of labeled cells were in the T2-T5 segments. Since the SCG cells innervating these 2 end organs lie in close proximity to each other within the ganglion, these results suggest that a specific transfer of the virus to the preganglionic neurons occurred, possibly via a transsynaptic mechanism. Third, virally infected glial cells were rarely found in the SCG or spinal cord. In summary, Bartha's K strain of PRV is a specific transneuronal retrograde grader marker in the sympathetic nervous system.