The endothelin (ET)-B receptor subtype is expressed on vascular endothelial and smooth muscle cells and participates in vasodilatation and vasoconstriction. Controversy exists regarding the role of the ET-B receptor as a mediator of systemic, pulmonary, and renal vasoconstriction in states of marked ET-1 activation. Moreover, the potential activation of endogenous ET-1 with secondary stimulation of the ET-A receptor in response to sarafotoxin S6c (S6c) remains unclear. This study was designed to assess the cardiovascular actions of ET-B activation with S6c in the presence and absence of selective ET-A antagonism with FR-139317 and dual ET-A/ET-B antagonism with SB-209670 in the anesthetized dog. Compared with time control (n = 5), S6c increased from baseline systemic vascular resistance (SVR) [28 ± 7 vs. 14 ± 3 resistance units (RU), P < 0.05] and pulmonary vascular resistance (PVR) (3.2 ± 0.7 vs. 0.9 ± 0.3 RU, P < 0.05) and decreased cardiac output (CO) (1.7 ± 0.3 vs. 0.5 ± 0.1 l/min, P < 0.05), with no differences in renal vascular resistance in association with increases in plasma ET-1. S6c also decreased mixed venous oxygen saturation (Sv_O2) (56 ± 6 vs. 76 ± 5%, P < 0.05). Selective ET-A receptor antagonism did not affect the actions of S6c, with the exception that ET-A receptor antagonism blocked the increase in SVR to high-dose S6c. Dual ET-A/ET-B receptor antagonism attenuated the increase from baseline in SVR (7 ± 1 vs. 28 ± 7 RU, P < 0.05) and PVR (0.7 ± 0.2 vs. 3.2 ± 0.7 RU, P < 0.05) and decrease from baseline in CO (0.9 ± 0.1 vs. 1.7 ± 0.3 l/min, P < 0.05) and Sv_O2 (7 ± 3 vs. 20 ± 3%, P < 0.05) observed with S6c alone. In summary, this study demonstrates an important role of ET-B receptor activation in vivo, which results in increases in plasma ET-1 and systemic and pulmonary vasoconstriction and reductions in CO and Sv_O2. This study also supports a modest role for the ET-A receptor in mediating the systemic vasoconstrictor response to high-dose S6c.