The orientation of human erythrocyte membrane protein was examined by enzymic iodination using lactoperoxidase with the glucose-oxidase system for generating peroxide, followed by proteolytic digestion. The outer surface of intact cells was labeled with 125I and the cytoplasmic surface of either resealed ghosts containing lactoperoxidase or of inside-out vesicles was labeled with 131I. Following iodination, the outer surface (resealed ghosts) or the cytoplasmic surface (outer surface of inside-out vesicles) was digested with trypsin, chymotrypsin, or pronase. Sodium dodecyl sulfate gel electrophoresis of the isolated membranes revealed three major and several minor peaks of radioactivity. Their surface orientation, defined within the limits of the specificity of the probes used, was as follows: the three major peaks consist of: (a) a 90,000 to 100,000 molecular weight component labeled on both surfaces; its proteolytic digestion profile indicated that it spans the membrane in an asymmetric manner and that it is composed of more than one peptide; (b) the major red cell membrane glycoprotein (apparent molecular weight 60,000) which is labeled and digested at only the outer surface; and (c) peptide(s) of high molecular weight (approximately 200,000), labeled and digested at only the cytoplasmic surface. The minor components include a glycoprotein of approximately 25,000 (apparent molecular weight) accessible to both surfaces and peptides of 60,000 to 70,000, 45,000, and 20,000 molecular weight labeled only on the inner surface.