Antiserum against rat adipocyte intrinsic plasma membrane proteins raised in rabbits has previously been shown to mimic insulin action on rat adipocyte glucose transport activity and lipolysis. In the present studies, extraction of lipids from adipocyte membranes previously treated with dimethylmaleic anhydride to remove extrinsic proteins resulted in no loss of antigenic activity indicating that a lipid or glycolipid is not a major antigenic determinant in the intrinsic membrane protein preparation. Periodate treatment of adipocyte membranes abolished subsequent antigen-antibody interaction. Immobilized wheat germ agglutinin or concanavalin A adsorbed all of the antigenic components of detergent-solubilized adipocyte intrinsic membrane proteins and these membrane antigens were released upon incubation with N-acetyl-glucosamine or α-methyl-D-mannoside, respectively. The purified immunoglobulin fraction of antiserum against intrinsic membrane proteins was immobilized by reaction with CNBr-Sepharose to form an immunoaffinity column, and cholate-solubilized intrinsic membrane proteins were passed through the column. The 94,000 dalton glycoprotein fraction present in the original sample was adsorbed by the column, whereas cytochalasin B-sensitive D-glucose transport activity could be reconstituted from the material which did pass through the column. Elution of the column with 5 M NaI in order to release bound antigens produced a material enriched in the 94,000 dalton glycoprotein as well as several other proteins. 125I-Insulin was cross-linked to its receptor on the adipocyte plasma membrane as previously described. When such detergent-soluble material containing 125I-insulin receptor complexes was passed through immunoaffinity columns, little or no radioactivity was bound to the columns. These results indicate that the 94,000 dalton glycoprotein and several minor components of the adipocyte membrane represent the major antigens which react with the anti-intrinsic membrane protein antibodies produced in these studies. The results further demonstrate that the insulin-like activity of these antisera is not elicited by direct interaction of the antibodies with either the insulin receptor or the D-glucose transport system.