The location and distribution of viral RNA were examined in the CNS tissues of weanling mice acutely infected with the GDVII strain of Theiler''s murine encephalomyelitis virus. Viral RNA was detected by autoradiography following in situ hybridization with a viral RNA-specific 3H-labeled complementary DNA synthesized in vitro. Viral RNA was detected in pyramidal neurons of the hippocampus, cerebral cortex, brainstem nuclei, thalamus, basal ganglia and spinal cord. Autoradiographic grains could be detected in the axonal and dendritic processes of many infected neurons. No viral RNA was detected in any cell of the cerebellum or white matter. In addition to demonstrating the location of viral RNA in infected CNS tissues and hence the sites of viral replication during this acute polioencephalomyelitis, necrosis of hippocampal neurons appears to be due to lytic infection, rather than to hypoxia.