We have utilized the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor (IR). We identified a human cDNA encoding a protein that appears to be the human homolog of the yeast MAD2 protein, which we term hMAD2. The yeast MAD2 protein was first identified in a genetic screen to identify cell cycle checkpoint regulatory proteins, yet the mechanism by which MAD2 functions in cell cycle control is currently unclear. Here we show that hMAD2 requires the COOH-terminal 30 amino acids of the IR for interaction and that hMAD2 does not interact with the related insulin-like growth factor I receptor. Interestingly, hMAD2 does not require IR tyrosine autophosphorylation for interaction because it interacts with a kinase-dead IR in the yeast two-hybrid system. In support of this finding, hMAD2-GST fusions were found to interact strongly in vitro with receptors derived from noninsulin-stimulated cells. Furthermore, using two independent in vitro assays, IR activation was found to significantly reduce the interaction of hMAD2 with the IR. Lastly, we show that hMAD2 can be coimmunoprecipitated with the IR from Chinese hamster ovary IR cell lysates, suggesting that this interaction occurs in vivo in cells of mammalian origin. Our results suggest that hMAD2 represents a novel class of proteins that is specific for interaction with the IR as compared with the insulin-like growth factor I receptor and that interacts best with the inactive IR and is released upon receptor autophosphorylation. The function of hMAD2 and its potential role in insulin signaling remain to be elucidated.