The cysteine residues involved in the class I disulfide bonds between the alpha subunits in the (alpha beta)2 dimer of the human insulin receptor have been identified by labeling with N-ethylmaleimide and by site-directed mutagenesis. Both cysteine 524 and cysteine 682 form interchain disulfide bonds; their conversion to serine residues results in the absence of receptor dimers and the presence of alpha beta monomers. The receptor monomers have a slightly lower affinity for insulin than the native receptor dimers. Insulin binding to the receptor monomers promotes their dimerization in the plasma membrane; at nanomolar concentrations of receptor, both unliganded and liganded receptors are monomers. Receptor monomers are stimulated by insulin to autophosphorylate and to phosphorylate exogenous subtrates with the same efficiency as the receptor dimers. The conclusion is that receptor dimerization is not required to activate the tyrosine kinase activity of the insulin receptor.