In two cohorts of 174 and 165 obese Caucasian children, we measured insulin sensitivity and genotyped insulin receptor substrate IRS-1 and IRS-2 genes for the Arg972Gly and the Asp1057Gly variants, respectively. Because IRS-1 and IRS-2 have complementary roles in insulin signaling, we classified the genotypes in three categories: those with none of the variants in IRS-1 or IRS-2, those with one variant in IRS-1 or IRS-2, and those with variants in both IRS-1 and 2 proteins. The obese children with either the IRS-1 or IRS-2 variant had a mean insulin sensitivity index (2.9 +/- 0.2 in cohort 1, 2.7 +/- 0.1 in cohort 2) only slightly lower than the children having no variant in either gene (3.1 +/- 0.2 and 3.5 +/- 0.3, respectively). However, patients having variant alleles in both IRS-1 and IRS-2 genes showed a 25-35% decrease in sensitivity (2.3 +/- 0.2 and 2.0 +/- 0.2, respectively) when compared with nonvariant homozygotes (P < 0.001). These observations are reminiscent of the insulin sensitivity phenotypes in double IRS-1(+/-) IRS-2(+/-) heterozygous knockout mice. Our results stress the need for combined genotype analysis when candidate genes are functionally involved in the same pathway.