Endothelin-1 (ET-1) affects glucose uptake in adipocytes and may play an important role in adipose physiology. One of the principal functions of adipose tissue is the provision of energy substrate through lipolysis. In the present study, we investigated the effects of ET-1 on lipolysis in 3T3-L1 adipocytes. When glycerol release into the culture medium was measured as an index of lipolysis, the results showed that ET-1 caused a significant increase, which was time- and dose-dependent. Using a concentration of 10 nM ET-1, stimulation of glycerol release plateaued after 4 h exposure. This effect was inhibited by the ET_A receptor antagonist, BQ-610 (10 µM), but not by the ET_B receptor antagonist, BQ-788 (10 µM). To further explore the underlying mechanisms of ET-1 action, we examined the involvement of the cAMP-dependent protein kinase A-mediated, phospholipase A2 (PLA2)-mediated, protein kinase C (PKC)-mediated, phosphatidylinositol 3 (PI3)-kinase-mediated, and the mitogen-activated protein kinase (MAPK)-mediated pathways. Inhibition of adenylyl cyclase activation by SQ22536 (100 µM) did not block ET-1-induced lipolysis. Pretreatment of adipocytes with the PLA2 inhibitor, dexamethasone (100 nM), the PKC inhibitor, H7 (6 µM), or the PI3-kinase inhibitor, wortmannin (100 nM), also had no effect. ET-1-induced lipolysis was blocked by inhibition of ERK activation using PD98059 (75 µM), whereas a p38MAPK inhibitor (SB203580; 20 µM) had no effect. Results of Western blot further demonstrated that ET-1 induced ERK phosphorylation. These data show that ET-1 induces lipolysis in 3T3-L1 adipocytes via a pathway that is different from the conventional cAMP-dependent pathway used by isoproterenol and which involves ERK activation.