We searched for the presence of human immunodeficiency virus (HIV) in fresh-frozen muscle biopsy specimens from 10 patients with HIV-associated polymyositis (HIV-PM) using (a) 35S-labeled HIV-RNA transcript of the virus and in situ hybridization, and (b) polymerase chain reaction and slot-blot hybridization utilizing primers amplifying sequences from the gag and pol genes of the HIV genome. With in situ hybridization, positive signals were detected in sparse lymphoid cells surrounding the muscle fibers, but not within the muscle fibers, in up to two consecutive sections in 6 of the 10 specimens. By the polymerase chain reaction, amplified HIV-specific sequences were noted in 2 specimens, but in only 2 of 8 consecutive sections, implying infection of lymphoid cells rather than muscle fibers. Muscle cultures from six specimens failed to show integrated HIV sequences within the myotubes. We conclude that HIV sequences or transcriptional products are not present within the muscle fibers or the cultured myotubes of patients with HIV-PM. This indicates that: (a) viral replication does not take place within the muscle; (b) integration of HIV proviral genome does not occur within the myonuclei or satellite cells; and (c) HIV-PM does not seem to be due to a persistent infection of the muscle fiber by the virus.