There are strong correlations between impaired insulin-stimulated glucose metabolism and increased intramuscular lipid pools; however, the mechanism by which lipids interact with glucose metabolism is not completely understood. Long-chain acyl CoAs have been reported to allosterically inhibit liver glucokinase (hexokinase IV). The aim of the present study was to determine whether long-chain acyl CoAs inhibit hexokinase in rat and human skeletal muscle. At subsaturating glucose concentrations, 10 micromol/l of the three major long-chain acyl-CoA species in skeletal muscle, palmitoyl CoA (16: 0), oleoyl CoA (18: 1, n= 9), and linoleoyl CoA (18: 2, n= 6), reduced hexokinase activity of rat skeletal muscle to 61+/-3, 66+/-7, and 57+/-5% of control activity (P< 0.005), respectively. The inhibition was concentration-dependent (P< 0.005) with 5 pmol/l producing near maximal inhibition. Human skeletal muscle hexokinase was also inhibited by long-chain acyl CoAs (5 pmol/l palmitoyl CoA decreased activity to 75+/-6% of control activity, P< 0.005). Inhibition of hexokinase in rat and human muscle by long-chain acyl CoAs was additive to the inhibition of hexokinase by glucose-6-phosphate (an allosteric inhibitor of hexokinase). This inhibition of skeletal muscle hexokinase by long-chain acyl CoA suggests that increases in intramuscular lipid metabolites could interact directly with insulin-mediated glucose metabolism in vivo by decreasing the rate of glucose phosphorylation and decreasing glucose-6-phosphate concentrations.