Long-chain fatty acids (LCFA) derived from lipolysis in adipose tissue, from lipolysis of circulating VL DL-triacylglycerol and from lipolysis of intramyocellular triacylglycerol (IMTG) serve important functions in skeletal muscle. Apart from the fundamental role as a fuel for energy production, LCFA serve as important components of biological membranes and in selected signal tranduction pathways to alter gene expression. LCFA from the circulation are either immediately oxidized or esterified to triacylglycerol (TG), which is stored in lipid droplets (LDs), recognized as functional organelles consisting of a core of TG and cholesteryl esters surrounded by a phosholipid monolayer and with several regulatory proteins associated with the LD surface. The mobilization of fatty acids from the IMTG pool are catalyzed by three lipases: adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), and monoacylglycerol lipase (MAGL) which sequentially degrade TG, diacylglycerol (DAG) and monoacylglycerol (MAG), respectively. The specific activity of ATGL for TG is 10 times higher than that for DAG, while HSL preferentially hydrolyses DAG with a 10-times higher lipolytic rate compared to TG. In skeletal muscle HSL is considered the primary lipase activated by contractions This notion is based on in vitro activity measurements, where the contraction induced increase in TG-lipase activity was completely blocked when adding an HSL-antibody to the assay medium. However, the in vitro activity assay does not include changes in important regulatory events such as translocation of lipases to the lipid droplets and interaction with lipid droplet associated proteins, and therefore may not entirely reflect the acute activation of muscle TG-lipases in vivo. In addition, in several human studies dissociations between in vitro HSL activity and net change in IMTG content during exercise have been observed. This may reflect that lipases other than HSL are at play. Knowledge about ATGL in skeletal muscle is limited, but ATGL protein expression and activity have been demonstrated in both rodent and human skeletal muscle. The functional importance of ATGL for basal TG-hydrolysis in skeletal muscle is highlighted by the finding of massive IMTG-accumulation in ATGL-KO mice. These data strongly suggest that ATGL has an important role for skeletal muscle TG-hydrolysis. However, it is not known under which physiological conditions ATGL is activated in skeletal muscle. Dysregulation of lipid metabolism in skeletal muscle is related to several pathological disorders, among these insulin resistance. Lipid intermediates or metabolic by-products derived from esterification of fatty acids into IMTG, from lipolysis of IMTG or from β-oxidation are all candidates involved in insulin resistance. Several molecular mechanisms behind lipid induced insulin resistance in skeletal muscle have been suggested, however this topic is still unresolved.