Casein kinase II activities were purified from human erythrocyte membrane and cytosolic fractions to apparent homogeneity. The kinases isolated from the membrane and cytosolic fractions exhibited the same subunit composition and the ability to utilize ATP and GTP as phosphoryl donors. Antibodies against the alpha and alpha' subunits of human casein kinase II cross reacted with the corresponding subunits of both erythrocyte casein kinases. Spermine, spermidine, putrescine, and polylysine stimulated to varying degrees the activities of erythrocyte casein kinase II, whereas heparin inhibited the kinase activities. Both kinases were found to catalyze the phosphorylation of several erythrocyte membrane cytoskeletal proteins, including spectrin, ankyrin, adducin, protein 4.1, and protein 4.9. Unlike casein kinase I, casein kinase II did not phosphorylate band 3 appreciably. A preliminary estimate indicates that both human erythrocyte membrane and cytosolic casein kinase II catalyze the incorporation of approximately 1.2 and 3.5 moles of phosphate into each mole of spectrin and ankyrin, respectively. An analysis of the phosphopeptide maps of ankyrin indicates that both membrane and cytosolic kinases phosphorylate the same domains within ankyrin. These data, taken together, suggest that the type II casein kinases isolated from human erythrocyte membrane and cytosol are either identical or closely related and may play a role in the regulation of cytoskeletal protein interactions.