Isolated myocytes were prepared from the adult rat heart and characterized for viability. The myocytes were exposed to 125I-insulin, and the 125I-insulin-receptor complex was extracted with 1% Triton X-102 and then applied to a DEAE-Sephacel column. When the 125I-insulin-receptor fractions from the ion exchange chromatography were applied to a Sepharose CL-6B column, a 140,000-dalton complex with high specific radioactivity was found. Alternatively, when myocyte insulin receptors were first extracted with 1% Triton X-102 without prior exposure to 125I-insulin and then applied to a DEAE-Sephacel column, three peak protein fractions were obtained. They were treated separately with 125I-insulin and the 125I-insulin-protein complexes were covalently cross-linked with disuccinimidyl suberate. The cross-linked samples were applied to a Sepharose CL-6B column and the radioactive protein fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first and second peak samples from the ion exchange chromatography yielded complexes of low specific radioactivity, which appear to be formed by nonspecific random binding of 125I-insulin to the solubilized membrane proteins. In contrast, Sepharose CL-6B gel filtration of the cross-linked sample from the third peak fractions gave a major highly radioactive 125I-insulin-receptor complex with a molecular weight of 370,000 and a minor complex of low radioactivity with a molecular weight of 140,000. Upon sodium dodecyl sulfate-gel electrophoresis, the 370,000-dalton complex was dissociated to 130,000- and 82,000-dalton components and the 140,000-dalton complex was dissociated to a 47,000-dalton component.