To investigate whether this could be due to sex-specific regulation of hormone-sensitive lipase (HSL) and to use sex comparison as a model to gain further insight into HSL regulation, nine women and eight men performed bicycle exercise (90 min, 60% V O2 peak), and skeletal muscle HSL expression, phosphorylation, and activity were determined. Supporting previous findings, basal IMTG content (P Ͻ 0.001) and net IMTG decrease during exercise (P Ͻ 0.01) were higher in women than in men and correlated significantly (r ϭ 0.72, P ϭ 0.001). Muscle HSL mRNA (80%, P ϭ 0.11) and protein content (50%, P Ͻ 0.05) were higher in women than in men. HSL total activity increased during exercise (47%, P Ͻ 0.05) but did not differ between sexes. Accordingly, HSL specific activity (HSL activity per HSL protein content) increased during exercise (62%, P Ͻ 0.05) and was generally higher in men than in women (82%, P Ͻ 0.05). A similar pattern was observed for HSL Ser 659 phosphorylation, suggesting a role in regulation of HSL activity. Likewise, plasma epinephrine increased during exercise (P Ͻ 0.05) and was higher in men than in women during the end of the exercise bout (P Ͻ 0.05). We conclude that, although HSL expression and Ser 659 phosphorylation in skeletal muscle during exercise is sex specific, total muscle HSL activity measured in vitro was similar between sexes. The higher basal IMTG content in women compared with men is therefore the best candidate to explain the higher IMTG net hydrolysis during exercise in women. intramuscular triacylglycerol; epinephrine; extracellular signal-regulated kinase TRIACYLGLYCEROL STORED IN skeletal muscle fibers (intramuscular triacylglycerol, IMTG) represents a large source of energy that may be used for muscle contraction during exercise. In men, it is still controversial to what extent IMTG is utilized during exercise (18,29,40). This is probably so because methodological limitations in measuring IMTG content have made it difficult to detect the relatively small net hydrolysis of IMTG that appears to occur during submaximal exercise in men (13,29,40,46). On the other hand, in women it has been shown that IMTG content is reduced by ϳ25% during 90-min bicycle exercise at 60% peak oxygen uptake (V O 2 peak ) (33), and it can be estimated that IMTG covers a large fraction (ϳ25%) of oxidative energy production in such an exercise bout in women (27). Therefore, the degree of IMTG hydrolysis during submaximal exercise appears to depend on sex, being higher in women than in men. It is presently unknown what may be the cause underlying this sex difference in IMTG hydrolysis during exercise.Hormone-sensitive lipase (HSL) is thought to catalyze the hydrolysis of IMTG in skeletal muscle as it does in adipose tissue (14,21). In rodent as well as in human skeletal muscle, neutral lipase activity increases during contraction (22,28,41), and it has been shown that the increase in neutral lipase activity elicited by contraction is completely accounted for by HSL activation both in rats and in man (22,28,45)...