Mobilization of fatty acids from stored triacylglycerol (TG) in adipose tissue and skeletal muscle (intramyocellular triacylglycerol - IMTG) requires activity of lipases. Although exercise training increases the lipolytic capacity of skeletal muscle, the expression of hormone sensitive lipase (HSL) is not changed. Recently, adipose triglyceride lipase (ATGL) was identified as a TG specific lipase in various rodent tissues. To investigate whether human skeletal muscle ATGL protein is regulated by endurance exercise training, 10 healthy young men completed 8 weeks of supervised endurance exercise training. Western blotting analysis on lysates of skeletal muscle biopsy samples revealed that exercise training induced a 2-fold increase in skeletal muscle ATGL protein content. In contrast to ATGL, expression of comparative gene identification 58 (CGI-58), the activating protein of ATGL, and HSL protein was not significantly changed after the training period. The IMTG concentration was significantly decreased by 28% at termination of the training program compared to before. HSL-phoshorylation at Ser660 was increased, HSL- Ser659 phosporylation was unchanged and HSL-phoshorylation at Ser565 was decreased altogether indicating an enhanced basal activity of this lipase. No change was found in the expression of diacylglycerol acyl transferase 1 (DGAT1) after training. Inhibition of HSL with a mono-specific, small molecule inhibitor (76-0079) and stimulation of ATGL with CGI-58 revealed that a significant ATGL activity is present in human skeletal muscle. These results suggest that ATGL in addition to HSL may be important for human skeletal muscle lipolysis.