We have developed an enzyme-linked immunosorbent assay (ELISA) specific for autoantibodies directed against the autoantigen Jo-1 (histidyl-tRNA synthetase) using antigen prepared biochemically from HeLa cells. No other patient sera, including those containing antibodies directed at threonyl-tRNA synthetase or alanyl-tRNA synthetase, reacted in the assay. Screening of sera from 169 patients with a variety of autoimmune and neuromuscular diseases confirmed that anti-Jo-1 antibodies are confined to a subgroup of patients with pure polymyositis, pure dermatomyositis, or myositis associated with another rheumatic disease.