Picornaviruses can initiate chronic inflammation that persists after the virus can no longer be cultured from inflamed tissues. In an attempt to understand this transition we have sought evidence for viral persistence by methods that detect viral genome independent of whether or not whole competent virus is present. In mice infected with a myotropic variant of encephalomyocarditis virus, EMC-221A, virus can be cultured in high yield at 1 wk and in low yield at 2 wk from skeletal muscle, heart, and brain; a small number of plaque-forming units could be cultured from brain at 4 wk. By contrast, in situ hybridization detected viral nucleic acid at least a week or two thereafter, often in single cells. In the skeletal muscle, inflammation disappeared by 3 wk, but in heart it remained for the full 12 wk of observation. In the brain, microglial nodules, sometimes with associated viral nucleic acid, were present for a long period. Application of this technique allows a more accurate assessment of the role of viral persistence in the pathogenesis of virus-initiated but apparently autoimmune inflammation.