Little is known about the regulation of catecholamine-stimulated lipolysis in human skeletal muscle. Therefore, β-adrenergic regulation of lipolysis and blood flow was investigated in healthy subjects in vivo by use of microdialysis of the gastrocnemius muscle. First, during a hypoglycemic, hyperinsulinemic clamp, which induces a lipolytic response in skeletal muscle tissue, the muscle was locally perfused with β-adrenoceptor blocking agents. Perfusion with nonselective (propranolol) and β2-selective (ICI-118551) blocking agents counteracted the hypoglycemia-induced lipolysis ( P < 0.01), but perfusion with metoprolol (β1-blocker) did not affect the glycerol response. Second, selective β-adrenoceptor agonists were perfused in situ into skeletal muscle during resting conditions. β2-Adrenoceptor stimulation with terbutaline induced a concentration-dependent increase in skeletal muscle glycerol levels and in tissue blood flow, whereas perfusion with β1- or β3-adrenoceptor agonists (dobutamine or CGP-12177) did not influence the glycerol concentration or blood flow. In conclusion, in skeletal muscle tissue, only the β2-subtype is of importance among β-adrenoceptors for regulation of lipolysis and blood flow. This is in contrast to adipose tissue, where β1- and β3-adrenoceptors are also involved.