The noncovalently associated 125I-insulin-receptor complex was isolated from human erythrocyte membranes after allowing 125I-insulin to interact with the membranes followed by extraction of the 125I-insulin-receptor complex with Triton X-102 or, alternatively, by complete solubilization of the membranes with sodium dodecyl sulfate (SDS), removal of SDS, and then treatment of the solubilized sample with 125I-insulin. Sepharose CL-6B column chromatography of the 125I-insulin-receptor complex obtained by both of the above procedures yielded a highly radioactive 140,000-Da complex which was dissociated into small peptides when subjected to SDS-polyacrylamide gel electrophoresis. In contrast, when the 125I-insulin-treated membrane sample was extracted with Triton X-102, purified by DEAE-Sephacel ion exchange chromatography, covalently cross-linked with disuccinimidyl suberate, and then subjected to SDS-polyacrylamide gel electrophoresis, a highly radioactive component with M(r) = 53,000 was obtained. On the other hand, when the Triton X-102-solubilized membrane receptor sample was fractionated by DEAE-Sephacel ion exchange chromatography, complexed with 125I-insulin, covalently cross-linked, and then applied to a Sepharose CL-6B column, a 95,000-Da complex with high specific radioactivity was obtained. Upon SDS-polyacrylamide gel electrophoresis, the 95,000-Da complex was dissociated into a 53,000-Da component which appeared identical with that obtained from the receptor complex described above which was obtained by direct interaction of the membranes with 125I-insulin.