Histidyl-tRNA synthetase is purified to near homogeneity from rat liver. The subunit molecular weight of histidyl-tRNA synthetase is 50,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The Stokes radius and the sedimentation coefficient of histidyl-tRNA synthetase are 38 A and 6.0 S, respectively. The native molecular weight of histidyl-tRNA synthetase is calculated to be 96,000 on the basis of its hydrodynamic properties. The purified histidyl-tRNA synthetase reacts with the myositis-specific anti-Jo-1 antibodies. Anti-Jo-1 immunoglobulin G reacts with the native form of histidyl-tRNA synthetase and does not react or only weakly reacts with the denatured form. The anti-Jo-1 antibodies exhibit stronger inhibition toward histidyl-tRNA synthetase that has been preincubated with tRNA than that without preincubation. Anti-Jo-1 antibodies behave as a noncompetitive inhibitor with respect to tRNA in the aminoacylation reaction catalyzed by histidyl-tRNA synthetase. The structural features of the antigen of the anti-Jo-1 antibodies in light of these results are discussed.