Obesity and insulin resistance are related to both enlarged intramyocellular triacylglycerol stores and accumulation of lipid intermediates. We investigated how lipid overflow can change the oxidation of intramyocellular lipids (ICL(OX)) and intramyocellular lipid storage (ICL). These experiments were extended by comparing these processes in primary cultured myotubes established from healthy lean and obese type 2 diabetic (T2D) individuals, two extremes in a range of metabolic phenotypes. ICLs were prelabeled for 2 days with 100 microM [(14)C]oleic acid (OA). ICL(OX) was studied using a (14)CO(2) trapping system and measured under various conditions of extracellular OA (5 or 100 microM) and glucose (0.1 or 5.0 mM) and the absence or presence of mitochondrial uncoupling [carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)]. First, increased extracellular OA availability (5 vs. 100 microM) reduced ICL(OX) by 37%. No differences in total lipolysis were observed between low and high OA availability. Uncoupling with FCCP restored ICL(OX) to basal levels during high OA availability. Mitochondrial mass was positively related to ICL(OX), but only in myotubes from lean individuals. In all, a lower mitochondrial mass and lower ICL(OX) were related to a higher cell-associated OA accumulation. Second, myotubes established from obese T2D individuals showed reduced ICL(OX). ICL(OX) remained lower during uncoupling (P < 0.001), even with comparable mitochondrial mass, suggesting decreased mitochondrial function. Furthermore, the variation in ICL(OX) in vitro was significantly related to the in vivo fasting respiratory quotient of all subjects (P < 0.02). In conclusion, the rate of ICL(OX) is dependent on the availability of extracellular fatty acids and mitochondrial function rather than mitochondrial mass.