Our aim was to determine the importance of changes in phosphorylation of key insulin signaling intermediates in the insulin resistance observed in skeletal muscle of rats fed diets high in saturated or n-6 polyunsaturated fat. We used phospho-specific antibodies to measure the time-course of phosphorylation of key components of the insulin signaling pathway by immunoblotting, during the initial stages of a physiological elevation in circulating insulin concentration. The phosphorylation of insulin receptor at Tyr1162/1163 (IR Tyr1162/1163) increased over 20 min of insulin infusion while the downstream phosphorylation of insulin receptor substrate-1 Tyr612 (IRS-1 Tyr612) peaked at 5 min and declined thereafter. Interestingly, phosphorylation of IRS-1 at Tyr895 continued to increase over the 20 min time period and protein kinase B (PKB Ser473) phosphorylation reached a plateau by 5 min, demonstrating that different profiles of phosphorylation are involved in transmission of the insulin signal despite a constant level of insulin stimulation. In muscle from rats fed high n-6 polyunsaturated or saturated fat diets however, there was no insulin-stimulated increase in IRS-1 Tyr612 phosphorylation and a temporal difference in PKB Ser473 phosphorylation, despite no difference in IR Tyr1162/1163 phosphorylation, IRS-1 Tyr895 phosphorylation and extracellular signal-regulated kinase (ERK) phosphorylation. These results demonstrate that under conditions of increased insulin, similar to those used to assess insulin action in vivo, chronic high-fat feeding impairs insulin signal transduction related to glucose metabolism at the level of IRS-1 Tyr612 and PKB Ser473 and that these effects are independent of the type of fat used in the high-fat diet.