Antibodies to Jo-1 (alpha Jo-1) are most characteristically detected in patients with the idiopathic inflammatory muscle disease polymyositis (PM). The Jo-1 antigen has previously been identified as histidyl-tRNA synthetase (HRS). In order to clarify the cellular localization of the antigenic targets recognized by the alpha Jo-1 antibody, immunofluorescence (IF) studies were performed with cultured human myoblasts. Incubation with alpha Jo-1 positive sera demonstrated granular cytoplasmic as well as nuclear staining, but only the cytoplasmic fluorescence was specifically inhibited by preabsorbing the sera with recombinant histidyl-tRNA synthetase (rHRS). A polyclonal rabbit anti-rHRS sera demonstrated granular cytoplasmic IF which was also specifically inhibited by preincubation with rHRS protein. Alpha Jo-1 negative healthy control or patient sera demonstrated nonspecific low intensity staining. 35S methionine biosynthetically labelled myoblast cell extracts immunoprecipitated with alpha Jo-1 positive sera and analyzed by SDS-PAGE revealed a specific band of the same molecular weight as the rHRS antigen. Our studies demonstrate that alpha Jo-1 specifically binds to antigen in the cytoplasm of cultured myoblasts. Alpha Jo-1 has been shown to inhibit HRS activity in vitro. Given the importance of aminoacyl tRNA synthetases such as HRS to intracellular protein assembly, intracytoplasmic binding and enzyme inhibition in vivo may potentially contribute to the pathogenesis of autoimmune muscle damage in PM.