Antibodies to the insulin receptor were prepared in BALB/c mice by immunization with IM-9 human lymphocytes, a cell type that has a large number of plasma membrane insulin receptors. The spleens of these mice were then removed, and their lymphocytes were fused to a mouse myeloma cell line, FO cells. After screening over 1,200 resulting hybrids, one stable hybrid was obtained that produced IgG1 antibodies directed towards the insulin receptor. This antibody blocked 125I-labeled insulin binding to its receptor by more than 90% in three human tissues: IM-9 cultured lymphocytes, freshly isolated adipocytes, and placenta membranes. In contrast, the antibody did not inhibit insulin binding to rat adipocytes and rat liver plasma membranes, suggesting that the antibody was species specific. In IM-9 cells, which had their proteins prelabeled with [35S]methionine, the antibody precipitated two polypeptides with molecular weights of 135,000 and 95,000; these molecular weights are identical to those previously identified as the alpha and beta subunits of the insulin receptor. The monoclonal antibody inhibited the actions of insulin on both human adipocytes and fibroblasts, suggesting that the antibody was an antagonist of insulin action. The present studies suggest, therefore, that monoclonal antibodies to the insulin receptor may provide new insights into the structure of the insulin receptor and its interaction with insulin.