Insulin binding to the human insulin receptor (HIR) is characterized by negatively cooperative site-site interactions that give rise to a curvilinear Scatchard plot. Insulin binding to recombinant secreted HIRs is linear, suggesting that interactions between the transmembrane or cytoplasmic domains of the receptor heterodimers may be responsible for the generation of negative cooperativity. To determine the domains responsible, a series of HIR cDNAs encoding C-terminal deletion mutations was constructed; HIR.delta CT, HIR.delta TK, HIR.delta TMCP-encoded deletions of the tyrosine kinase regulatory, the tyrosine kinase regulatory and catalytic, the cytoplasmic and the transmembrane and cytoplasmic domains, respectively. When expressed in COS cells, all cDNAs were processed to mature alpha- and beta- subunits. The affinity of HIR.delta CT, HIR.delta TK, and HIR.delta CP for insulin were 2- to 3-fold greater than that of wild type HIR (HIR.WT) which was 4- to 5-fold greater than that of HIR.delta TMCP. Scatchard plots of HIR.delta CT, HIR.delta TK, and HIR.delta CP, like that of HIR.WT, were curvilinear. In contrast, that of HIR.delta TMCP was linear. We conclude that constraints imposed on HIR structure by membrane insertion and/or interactions between receptor transmembrane domains are essential for the generation of negative cooperativity. Further, interactions between the C-terminal regions of the cytoplasmic domains appear to modulate affinity for insulin.