We have previously shown that glycogen synthesis is reduced in lipid-treated C₂C₁₂ skeletal muscle myotubes and that this is independent of changes in glucose uptake. Here, we tested whether mitochondrial metabolism of these lipids is necessary for this inhibition and whether the activation of specific protein kinase C (PKC) isoforms is involved. C₂C₁₂ myotubes were pretreated with fatty acids and subsequently stimulated with insulin for the determination of glycogen synthesis. The carnitine palmitoyltransferase-1 inhibitor etomoxir, an inhibitor of -oxidation of acyl-CoA, did not protect against the inhibition of glycogen synthesis caused by the unsaturated fatty acid oleate. In addition, although oleate caused translocation, indicating activation, of individual PKC isoforms, inhibition of PKC by pharmacological agents or adenovirus-mediated overexpression of dominant negative PKC-, -, or - mutants was unable to prevent the inhibitory effects of oleate on glycogen synthesis. We conclude that neither mitochondrial lipid metabolism nor activation of PKC-, -, or - plays a role in the direct inhibition of glycogen synthesis by unsaturated fatty acids.