Mild treatment of human erythrocyte spectrin with trypsin produces discrete intermediate-sized peptides. The effects of buffer composition, enzyme-substrate ratio, temperature, and other experimental parameters on the resulting peptide pattern have been examined. Spectrin is capable of regaining its proteolytic resistance after NaDodSO4-induced denaturation, permitting the use of isolated subunits to study spectrin structure and function. Tryptic digestion of isolated subunits also has greatly facilitated the identification of the subunit origin of the intermediate-sized peptides. Isolated subunits could also be recombined to form functional units similar but not identical to the native dimeric form of the regions or domains connected by small protease sensitive segments. The structural integrity and accessibility of these sites is minimally affected by oligomeric state or proteolytic digestion conditions. The similarities of sizes, isoelectric points, and amino acid compositions of many intermediate-size peptides from areas of both subunits suggest that at least part of spectrin's structure may have evolved via replication of a single gene. A possible structural repeat of approximately 50,000 daltons is hypothesized.