Two molecular defects involving the spectrin heterodimer (SpD) contact site of the α chain (the αI domain) were previously identified using limited tryptic digestion followed by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both are characterized by atypical peptide maps which reveal a marked decrease of the 80,000-dalton αI domain and a formation of new major peptides of either 74,000 (Sp α(I/74)) or 46,000 (Sp α(I/46)) daltons. We now report a third variant of the spectrin α chain, designated sp α(I/65), in three unrelated black families. In all three probands, the percentage of SpD in the low ionic strength (0° C) membrane extracts was increased to 19% to 32%. One- and two-dimensional electrophoretic separations of limited tryptic digests of spectrin from all three probands revealed a decrease of the αI domain of spectrin and the concomitant appearance ranging from 5.2 to 5.3. The abnormal 65,000-dalton peptides could be stained with an antiserum which had been raised against the αI domain, indicating that it was derived from the αI domain.