CONTEXT: The obese insulin-resistant state is characterized by elevated lipid storage in skeletal muscle tissue. OBJECTIVE: We tested whether differences in muscle triacylglycerol (TAG) and diacylglycerol (DAG) lipase content and activity are associated with incomplete in vivo lipolysis and lipid accumulation. DESIGN AND PATIENTS: Two case-control studies were conducted on skeletal muscle biopsies from lean (n=13) and obese (n=10) men (study 1) and from 11 nonobese type 2 diabetic (T2D), obese T2D, and healthy normoglycemic men (study 2). MAIN OUTCOME MEASURES: Skeletal muscle lipase protein content and activity and muscle lipid content (TAG and DAG) were determined. RESULTS: Skeletal muscle hormone-sensitive lipase protein content was lower (0.39±0.07 vs. 1.00±0.19 arbitrary units; P=0.004) and adipose triglyceride lipase protein content was higher in obese men compared with lean controls (2.17±0.40 vs. 0.42±0.23 arbitrary units; P=0.008). This apparent difference in lipase content was accompanied by a 60% lower ratio of DAG to TAG hydrolase activity in the obese men (11.4±2.3 vs. 26.5±7.3 nmol/h*mg; P=0.045), implying incomplete lipolysis. Lower hormone-sensitive lipase and higher adipose triglyceride lipase content was confined to obesity per se, because it was observed solely in obese T2D men but not in healthy normoglycemic controls and nonobese T2D men. Muscle total DAG content was not higher in obese men but was even lower (6.2±0.7 vs. 9.4±0.9 μmol/mg dry weight; P=0.017). TAG content did not differ between groups (84.7±18.9 vs. 70.4±12.4 μmol/mg dry weight; P=0.543). CONCLUSIONS: Our data do not support an important role of total muscle DAG content in the development of insulin resistance in obese men.