Senescent cell antigen is a glycosylated polypeptide, migrating in the band 4.5 region of NaDodSO4/polyacrylamide gels, that appears on the surface of senescent and damaged cells. Appearance of the senescent cell antigen initiates specific binding of IgG autoantibodies to it and the removal of erythrocytes (RBCs). Previous experiments suggested that the senescent cell antigen may be immunologically related to an integral membrane protein designated band 3 that is involved in anion transport across the RBC membrane. In the present studies, senescent cell antigen was mapped along the band 3 molecule by using topographically defined fragments of band 3. Both binding of IgG eluted from senescent RBCs ("senescent cell IgG") to defined proteolytic fragments of band 3 in immunoblots and two-dimensional peptide mapping of senescent cell antigen, band 3, and defined proteolytic fragments of band 3 were used to localize senescent cell antigen along the band 3 molecule. The data suggest that the antigenic determinants of the senescent cell antigen that are recognized by physiologic IgG autoantibodies reside on an external portion of a naturally occurring transmembrane fragment of band 3 that has lost a Mr approximately equal to 40,000 cytoplasmic (NH2-terminal) segment and part of the anion-transport region. A critical cell-age-specific cleavage of band 3 appears to occur in the transmembrane, anion-transport region of band 3.