Accumulation of intramuscular long-chain acyl-CoA esters (LCACoA) has previously in animal and human models been suggested to play an important role in lipid induced insulin resistance. The aim of this study was to examine whether myotubes established from type 2 diabetic (T2D) subjects and lean controls express differences in long-chain acyl-CoA esters (LCACoA) precultured under physiological conditions and during chronic exposure to palmitate (PA) and oleic acids (OA) with/without acute insulin stimulation. No significant differences were found between diabetic and control myotubes, neither in the total amount nor among individual LCA-CoA species during basal and acute insulin stimulation. LCA-CoA accumulated during exposure to palmitic acid but not during exposure to oleic acid. During PA and OA exposure, only palmitoyl-CoA, oleoyl-CoA and total LCA-CoA change. PA exposure increased the palmitoyl-CoA, whereas oleoyl-CoA was reduced and vice versa during OA exposure. No differences were found in the LCA-CoA level between T2D and control subjects, neither in the total amount nor in the individual specific LCA-CoA species during fatty acid exposure. Chronic (24 h), high PA, but not OA exposure induced insulin resistance at the level of glycogen synthesis in control subjects. These results indicate that (1) no primary defects are responsible for LCA-CoA accumulation in diabetic subjects; (2) LCA-CoA changes in vivo are partly adaptive to changes in the PA level and possibly other saturated fatty acids; and (3) PA induced insulin resistance may be mediated through an increased level of palmitoyl-CoA.