Insulin resistance is a major feature of noninsulin-dependent diabetes mellitus. This resistance appears to involve molecules, apart from the insulin receptor, that are capable of altering its function. Previously, we reported that dermal fibroblasts from a female patient with insulin resistance and noninsulin-dependent diabetes produced an inhibitor of insulin receptor tyrosine kinase activity. We have now studied inhibitors in fibroblasts from four additional patients (one male and three females) with severe insulin resistance. Although clinical features were diverse, these patients had in common normal fasting glucose values, with fasting and postprandial hyperinsulinemia. The fibroblast insulin receptor content was within the normal range, but both basal and insulin-stimulated tyrosine kinase activity in fibroblast extracts were markedly decreased compared to those in extracts of fibroblasts from nondiabetic subjects. Studies revealed that these fibroblasts contained a glycoprotein inhibitor of insulin receptor tyrosine kinase activity. This inhibitor was not found in extracts of either similar insulin-resistant patients with normal insulin receptors or insulin-resistant patients with insulin receptor abnormalities. The inhibitor was not adsorbed with antiserum to either tyrosine phosphatases or fetuin. These studies thus suggest that one or more unique inhibitors of insulin receptor tyrosine kinase are present in fibroblasts of certain patients with severe insulin resistance. The presence of insulin receptor tyrosine kinase inhibitors in target cells, therefore, may constitute a novel mechanism of postreceptor insulin resistance.