I mmunoglobulin G (IgG) autoantibodies of 20 patients with autoimmune hemolytic anemia (AHA) were used in immunoaffinity assays with surface-radioiodinated human red blood cells (RBCs), and detergent-solubilized products were analyzed by SDS-PAGE/autoradiography. Four membrane proteins were identified as candidate autoantigens: a nonglycosylated polypeptide with an apparent molecular mass of 34 kD (p34) that was expressed in all available RBC phenotypes except Rhnull but differed consistently in apparent molecular mass from the 32-kD Rh(D) polypeptide co-isolated by IgG allo-anti-D; a heterogenous 37-55-kD glycoprotein, also deficient in Rhnull RBCs, which disappeared after deglycosylation by N-glycanase, with the appearance of a sharp, new approximately 31-kD band distinct from p34 and from Rh(D) polypeptide; a approximately 100-kD major membrane glycoprotein identified by immunoblotting as the band 3 anion transporter; and glycophorin A (GPA), also confirmed by immunoblotting. GP37-55 was not seen in the absence of p34, and both proteins are likely to be members of the Rh family. Indeed, a 34-kD polypeptide band and 37-55-kD poly-disperse "smear," isolated concurrently from the same labeled RBCs by IgG allo-anti-e, were indistinguishable from their autoantibody-isolated counterparts and may well be the same protein identified at different epitopes by the auto- and allo-antibodies. Individual AHA patients' autoantibodies isolated p34 and gp37-55, alone or in combination with band 3 (nine cases); strong band 3 alone (five cases); and combinations of band 3 with GPA (six cases). The autoantibodies of three additional patients whose AHA had been induced by alpha-methyldopa also isolated p34 and gp37-55.