The ability to maintain skeletal muscle mass appears to be impaired in insulin resistant conditions that are characterised by muscle lipid accumulation, such as type 2 diabetes. The present study investigated the effect of acutely increasing lipid availability on muscle protein synthesis. Seven healthy young male volunteers underwent a 7 h intravenous infusion of L-[ring-2H5]phenylalanine on two randomised occasions combined with either 0.9% saline or 10% Intralipid at 100 mL/h. After a 4 h 'basal' period, a 21 g bolus of amino acids was administered and a 3 h euglycaemic hyperinsulinemic clamp was commenced ('fed' period). Muscle biopsies were obtained from the vastus lateralis at 1.5, 4, and 7 h. Lipid infusion reduced fed whole-body glucose disposal by 20%. Furthermore, whereas mixed muscle fractional synthetic rate increased from the basal to fed period during saline infusion by 2.2-fold, no change occurred during lipid infusion, despite similar circulating insulin and leucine concentrations. This 'anabolic resistance' to insulin and amino acids with lipid infusion was associated with a complete suppression of muscle 4E-BP1 phosphorylation. We propose that increased muscle lipid availability may contribute to anabolic resistance in insulin resistant conditions by impairing translation initiation.