OBJECTIVE Excess intramyocellular triacylglycerol (IMTG), found especially in obese women, is slowly metabolized and, therefore, prone to longer exposure to intracellular desaturases. Accordingly, it was hypothesized that IMTG content correlates inversely with IMTG fatty acid (FA) saturation in sedentary subjects. In addition, it was validated if IMTG palmitic acid is associated with insulin resistance as suggested earlier. METHODS Cross-sectional human study. METHODS In skeletal muscle biopsies, which were obtained from sedentary subjects (34 women, age 48+/-2 years (27 obese including 7 type 2 diabetes (T2DM), body mass index (BMI)=35.5+/-0.8 kg m(-2)) and 25 men, age 49+/-2 years (20 obese including 6 T2DM, BMI=35.8+/-0.8 kg m(-2))), IMTG FA composition was determined by gas-liquid chromatography after separation from phospholipids by thin-layer chromatography. RESULTS Independently of gender saturated FA correlated inversely with IMTG (P<0.001) and monounsaturated FA (P<0.001) including total unsaturation of FA (P<0.002) correlated positively with IMTG. Obese women exhibited lower total saturated FA (P<0.001) and palmitic acid (P<0.001) than obese men independent of IMTG, the latter of which, however, was increased twofold in obese women compared to obese men (P<0.001). Polyunsaturated and long-chain polyunsaturated FA did not correlate with IMTG. Palmitic acid correlated positively with insulin resistance (homeostasis insulin resistance index, P<0.05), fasting glucose (P<0.01) and glycosylated hemoglobin (P<0.002) both in univariate analysis and after correction for gender and IMTG. CONCLUSIONS IMTG content correlates inversely with IMTG saturated FA, potentially reflecting a low turnover of excess IMTG prone to in situ desaturation probably by the ubiquitous stearoyl-CoA desaturase-1. IMTG FA composition is gender specific and implicates on insulin sensitivity and glycemic control.