Four anti-erythrocyte autoantibody responses (anti-X, anti-HB, anti-HOL, and anti-I) that occur spontaneously in mice have been characterized with regard to antigenic specificities, predominant immunoglobulin class, and pathogenetic importance. Each autoantibody response exhibits specificity for an independent erythrocyte membrane autoantigen (X, HB, HOL, or I) or a soluble analogue (SEA-X or SEA-HB) present in the plasma. The anti-X response, unique to NZB mice, is directed to a normally exposed murine erythrocyte autoantigen, whereas the anti-HB response is directed to a cryptic erythrocyte autoantigen exposed by limited enzymatic cleavage of the membrane. The anti-I response also is directed to a cryptic but distinct autoantigen, and anti-HOL autoantibodies react with an erythrocyte autoantigen located at the cytoplasmic surface of the membrane. Analysis of the predominant immunoglobulin class of each of the autoantibodies has demonstrated that anti-HB and anti-I antibodies are predominantly of IgM class, whereas anti-X and anti-HOL antibodies are IgG immunoblobulins. Only anti-X and anti-HB autoantibodies are recovered from Coombs' positive erythrocytes from NZB mice and erythrocytes with surface C3 are detected only in NZB mice greater than 9 months of age. These data suggest that only the anti-X and anti-HB responses are pathogenetically implicated in the autoimmune hemolytic anemia of NZB mice.