In humans and animal models, increased lipid content of skeletal muscle is strongly associated with insulin resistance. However, it is unclear whether this accumulation is due to increased uptake or reduced utilization of fatty acids (FAs). We used ³H-R-bromopalmitate tracer to assess the contribution of tissue-specific changes in FA uptake to the lipid accumulation observed in tissues of insulin-resistant, high fat-fed rats (HFF) compared with control rats (CON) fed a standard diet. To study FA metabolism under different metabolic states, tracer was infused under basal conditions, during hyperinsulinemic-euglycemic clamp (low FA availability) or during the infusion of intralipid and heparin (high FA availability). FA clearance was significantly increased in the red gastrocnemius muscle of HFF under conditions of low (HFF = 10.4 ± 1.1; CON = 7.4 ± 0.5 ml · min⁻¹ · 100 g⁻¹; P < 0.05), basal (HFF = 8.3 ± 1.4; CON = 4.5 ± 0.7 ml · min⁻¹ · 100 g⁻¹; P < 0.01), and high (HFF = 7.0 ± 0.8; CON = 4.3 ± 0.5 ml · min⁻¹ · 100 g⁻¹; P < 0.05) FA levels. This indicates an adaptation by muscle for more efficient uptake of lipid. Associated with the enhanced efficiency of FA uptake, we observed increases in CD36/FA translocase mRNA expression (P < 0.01) and acyl-CoA synthetase activity (P < 0.02) in the same muscle. FA clearance into white adipose tissue was also increased in HFF when circulating FA were elevated, but there was little effect of the high-fat diet on hepatic FA uptake. In conclusion, insulin resistance induced by feeding rats a high-fat diet is associated with tissue-specific adaptations that enhance utilization of increased dietary lipid but could also contribute to the accumulation of intramuscular lipid with a detrimental effect on insulin action.