Several polymorphisms have been identified in the amino acid sequence of human insulin receptor substrate-1 (IRS-1). Some of the variant sequences have been reported to be increased in prevalence among patients with noninsulin-dependent diabetes mellitus (NIDDM). This observation led to the hypothesis that these amino acid substitutions may impair the function of IRS-1, thereby causing the insulin resistance seen in patients with NIDDM. To address this question, we have designed studies to evaluate the effects of three variant sequences identified in our laboratory: Gly819-->Arg, Gly972-->Arg, and Arg1221-->Cys. We constructed four IRS-1 expression vectors for transfection in COS-7 cells: wild-type, single mutant (Gly819-->Arg), double mutant (Gly819-->Arg; Gly972-->Arg), and triple mutant (Gly819-->Arg; Gly972-->Arg; Arg1221-->Cys) IRS-1. The mutations did not alter the level of expression or the extent of insulin receptor-mediated tyrosine phosphorylation of recombinant IRS-1. Moreover, the mutations did not lead to a detectable impairment in the association of recombinant IRS-1 with important downstream effectors, including the p85 subunit of phosphatidylinositol 3-kinase and growth factor receptor-binding protein-2. We conclude that these amino acid substitutions do not appear to cause a major defect in the function of IRS-1, as judged by our assays. However, this type of assay probably lacks the sensitivity to detect subtle functional defects. In light of the suggestive associations observed in epidemiological studies, it is premature to totally discard the hypothesis that variant sequences of IRS-1 may contribute to the pathogenesis of NIDDM. Nevertheless, our studies cannot be interpreted as lending support to that hypothesis.