I nsulin resistance in Pima Indians appears to result from a post-receptor impairment of insulin signal transduction that affects only some responses to insulin. To identify the primary lesion responsible for insulin resistance, we investigated the influence of insulin on ribosomal protein S6 kinase activities in skeletal muscle of insulin-sensitive and insulin-resistant nondiabetic Pima Indians during a 2-h hyperinsulinemic, euglycemic clamp. In sensitive subjects, S6 kinase activity was transiently activated fivefold over basal activity by 45 min of insulin infusion. Although basal activities in the two groups were similar, the response to insulin was delayed and restricted to about threefold over basal in subjects resistant to insulin. Two major S6 kinase activities in extracts of human muscle were resolved by chromatography on Mono Q. Peak 1, which accounted for basal activity owes to an enzyme antigenically related to the 90-kD S6 kinase II, a member of the rsk gene family. The major insulin-stimulated S6 kinase eluted as peak 2 and is antigenically related to a 70-kD S6 kinase. Our results show that insulin resistance impairs signaling to the 70-kD S6 kinase.