Abstract The involvement of cysteine 524 of the insulin receptor in an intermolecular (class I) disulfide bond between the two α-subunits was investigated using site-directed mutagenesis. The oligomeric structure of the mutated receptor was partially disrupted, although a significant portion of the receptor remained in its heterotetrameric form. Interestingly, the mutated insulin receptor heterotetramer was more susceptible than the wildtype receptor to reduction to heterodimers by low concentrations of dithiothreitol. Insulin binding to solubilized mutant receptors was normal and the mutant receptors had normal affinity for insulin, but insulin binding to cells expressing mutant insulin receptors displayed positive cooperativity. Cysteine 524 is most likely involved in a class I disulfide bond and receptors mutated at this site displayed unusual insulin binding properties only in the cellular enviroment.