To investigate the hypothesis that the inflammatory muscle diseases (IMD) polymyositis (PM) and dermatomyositis (DM) may be due to a chronic, persistent enterovirus (EV) infection we sought to determine the prevalence of these viruses in muscle tissue using both nested polymerase chain reaction (PCR) and dot-blot hybridization assays. Thirty-six frozen muscle biopsies from 32 adult cases of IMD and 42 biopsies from 36 control subjects with other neuromuscular disorders were studied. Primers for PCR were chosen to conserved regions of the S-untranslated region of the EV genome. Constitutive Ableson tyrosine kinase (ABL) mRNA was detected by PCR to confirm the integrity of muscle RNA extracts. The sensitivity of the EV PCR was determined to be 40-400 copies (12.5-125 ag) of synthetic EV RNA transcript against a background of 1 mu g of cellular RNA. The specificity was assessed using a range of enteroviral and unrelated viral isolates extracted from cell cultures. Of the 78 samples tested, ABL mRNA was successfully detected in all but four samples. The time the biopsies spent in ultracold storage (1-73 months) did not appear to influence the integrity of extracted RNA. When assayed for EV RNA by nested PCR, none of 29 IMD cases (i.e., 28 PM and 1 DM) nor sequential biopsies from 3 PM patients were found to be positive. All 42 control biopsies were also negative for EV RNA, These results were confirmed by dot-blot hybridization of RNA extracts for all 74 cases. The results of the present study do not support the hypothesis that persistent coxsackie or related EV infection is the cause of IMD, assuming that primer annealing sites of the 5'-untranslated region of the viral genome are not mutated or partially deleted in affected tissue.