Excessive energy intake leads to fat overload and the formation of lipotoxic compounds mainly derived from the saturated fatty add palmitate (PAL), thus promoting insulin resistance (IR) in skeletal muscle. N-3 polyunsaturated fatty acids (n - 3PUFA) may prevent lipotoxicity and IR. The purpose of this study was to examine the differential effects of n 3PUFA on fatty acid metabolism and insulin sensitivity in muscle cells. C2C12 myotubes were treated with 500 mu M of PAL without or with 50 mu M of alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) for 16h. PAL decreased insulin-dependent AKT activation and glucose uptake and increased the synthesis of ceramides and diglycerides (DG) derivatives, leading to protein kinase CO activation. EPA and DMA, but not ALA, prevented PAL-decreased AKT activation but glucose uptake was restored to control values by all n 3PUFA vs. PAL. Total DG and ceramide contents were decreased by all n 3PUFA, but only EPA and DMA increased PAL beta-oxidation, decreased PAL incorporation into DG and reduced protein kinase CO activation. EPA and DMA emerge as better candidates than ALA to improve fatty acid metabolism in skeletal muscle cells, notably via their ability to increase mitochondrial beta-oxidation.