Hereditary pyropoikilocytosis (HPP) is a severe hemolytic anemia in which an instability of the red cell membrane skeleton has been correlated with structural and functional defects of spectrin. We now report that 13 unrelated HPP subjects have approximately 30% less spectrin than normal as evidenced by a decreased spectrin/band 3 ratio. We also examine the role of spectrin degradation as an underlying cause of this partial spectrin deficiency. Our studies demonstrate that the reduced spectrin content of HPP red cells remains constant during in vivo aging of the cells in the peripheral blood, as well as during in vitro incubation. Furthermore, immunoblotting experiments using an affinity-purified antispectrin antibody indicate that there is no loss of spectrin during membrane preparation and also that neither whole HPP red cells nor ghosts nor cytosol contains any abnormal spectrin degradation products. These data suggest that spectrin is not degraded and that it is stable on the membrane of the circulating HPP red cell. In contrast, however, incubation of free spectrin with a lysate of nucleated erythroid precursor cells indicates that HPP alpha I/46 spectrin, but not HPP alpha I/74 spectrin, is more susceptible to proteolytic degradation than a control. These data imply that the decreased spectrin content of HPP is not due to a single defect but that a more complex mechanism is involved. In HPP Sp alpha I/46 subjects, an increased proteolytic degradation in bone marrow erythroid precursors of cytosolic spectrin, prior to its assembly on the membrane, could contribute toward the partial spectrin deficiency.