Release of glycerol and free fatty acids (FFA) was investigated in human skeletal muscle strips. In the basal state, glycerol and free fatty acids (FFA) were released at almost equimolar rates (0.3 nmol/ng tissue/90 min). A non-selective -adrenoceptor agonist, isoprenaline, caused a concentration dependent stimulation of glycerol release whereas FFA-release was unaffected. Basal and isoprenaline-induced glycerol release correlated positively with the age of the donors (r=0.5, P < 0.005) but not with their body mass index (P 0.4). Biochemical experiments with hormone sensitive lipase (HSL) showed first, that most enzyme activity was both in the cytosol and mitochondrial fraction, and second, that it constituted the common long and active form of the protein. Electron microscopy studies in rat skeletal muscle using labeled highly specific hormone-sensitive lipase (HSL) antibodies verified the cytosolic location of HSL and, furthermore, indicated an accumulation of HSL adjoining mitochondria. These results suggest that FFA produced in myocytes during catecholamine-induced lipolysis are retained by the muscle and therefore by inference reutilized. It is conceivable that efficient hydrolysis of acylglycerol by hormone-sensitive lipase located in the cytosol as well as near the mitochondria may facilitate mitochondrial FFA oxidation. In addition, muscle lipolysis activity increases during aging and may be independent of total body fat.