Erythrocyte protein phosphorylation was examined in membrane preparations of 25 patients with hereditary spherocytosis (HS). Reduced phosphorylation in substrate polypeptides was observed in 22 HS erythrocyte membranes for both splenectomized and nonsplenectomized patients. A reduction in labeling was also observed in a polypeptide which was labeled only in the presence of cAMP. No reduction was observed in membranes from immunologically acquired spherocytosis cells or from cells from patients with hereditary elliptocytosis. Heating membranes to 45 degrees C caused negligible inhibition of membrane protein phosphorylation, while heating to 50 degrees C extensively inhibited phosphorylation. Dephosphorylation of membrane protein that occurred in isolated membranes was not dependent upon cAMP.