In hereditary pyropoikilocytosis (HPP) the erythrocyte membrane skeleton exhibits mechanical instability that can be correlated to defective self-association of spectrin heterodimers. To detect structural changes in the functional domains of HPP spectrin we have examined the peptide pattern produced by limited tryptic digestion of spectrin extracts from two families that contain three HPP patients. Limited tryptic digestion of all three HPP patients revealed a similar and reproducible decrease in the staining intensity of an 80,000-, and 22,000-, and an 88,000-dalton polypeptide with a concomitant increase in a 74,000- and a 90,000-dalton polypeptide as compared with controls. Only changes in the 80,000-, and 74,000-, and 22,000-dalton polypeptides could be correlated to defective spectrin self-association and the amount of spectrin dimers in 0°C extracts of the HPP patients and their affected kindred. Similar results were obtained when the tryptic digests were analyzed by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the affected 74,000- and 80,000-dalton polypeptides focusing into multiple spots ranging in isoelectric point from 5.3-5.4. When HPP spectrin dimers and tetramers were separated and subjected to trypsin digestion, changes in the 80,000-, 74,000-, and 22,000-dalton polypeptides were found predominantly in the spectrin dimer pool. Similar results were obtained for spectrin from two of the probands' mother, whom we have identified as an HPP carrier. We conclude that these HPP patients contain a population of normal, (principally tetrameric) and mutant (principally dimeric) spectrin. The latter is characterized by a defective spectrin dimer self-association due to conformational changes that affect the 80,000-dalton domain.