Restricted tryptic digestion of erythrocyte spectrin at 4 degrees C followed by two-dimensional (isoelectric-focusing/sodium dodecyl sulfate) polyacrylamide electrophoresis yields highly reproducible maps of approximately 50 peptides with molecular weights between 80,000 and 12,000. Based on molecular weight and isoelectric point (pI), each unique alpha- and beta-subunit domain can be identified and compared with spectrin peptides from other individuals. The alpha-subunit of spectrin from 60 Caucasian donors contains a 46,000-mol-wt tryptic domain, called alpha II-T46, Type 1; more extensive tryptic digestion of this domain generates peptides with molecular weights of 35,000, 30,000, 25,000, and 16,000. Spectrin from 29 of 37 black donors representing 14 kindreds shows variation in the molecular weight and/or pI of peptides from the alpha II domain. In the most common form, Type 2, alpha II tryptic peptides are increased in molecular weight by 4,000, and the pI becomes more basic. Other alpha II variants are characterized by either the 4,000 increase in molecular weight (Type 3) or by the basic shift in pI (Type 4). When limit peptide maps of intermediate-sized tryptic and CNBr peptides from the alpha II-domain Types 1 and 2 are compared, a consistent alteration in the chromatographic mobility of one limit peptide is observed. Polymorphism in the alpha II subunit of spectrin did not itself produce anemia, nor did it appear to alter the expression of an underlying hereditary spherocytosis or elliptocytosis. In six family studies, the alpha II 46,000-mol-wt variations observed were consistent with Mendelian inheritance.