We investigated the functional importance and signal transduction pathways of endothelin (ET)-B receptors in mediating ET-1-induced vasoconstriction in pig skin. Skin vasoconstriction was studied by monitoring the perfusion pressure of isolated perfused pig skin flaps (6 × 16 cm) at a constant flow rate. Intra-arterial infusion of the ET_A/B receptor agonist ET-1, the ET_B receptor agonists sarafotoxin 6C (S6c) and BQ-3020, or the thromboxane A₂ mimetic U-46619 (n = 4 or 5) caused a concentration-dependent skin vasoconstriction. The vasoconstrictor potency of ET-1 (EC₅₀ 3.1 × 10⁹ M) was lower (P < 0.05) than that of S6c (EC₅₀ 1.8 × 10⁹ M) and similar to that of BQ-3020 (EC₅₀ 2.6 × 10⁹ M). The vasoconstrictor potency of ET-1, S6c, and BQ-3020 was at least 300-fold higher than that of U-46619 (EC₅₀ 0.9 × 10⁶ M). The skin vasoconstrictor effect of ET-1 (10⁹-10⁸ M) was partially inhibited by 10⁵ M BQ-123, an ET_A receptor antagonist. Further inhibition was achieved with the combination of 10⁵ M BQ-123 and BQ-788 (an ET_B receptor antagonist) or with an ET_A/B receptor antagonist (10⁵ M bosentan or PD-145065) (n = 5; P < 0.05). In addition, the skin vasoconstrictor effect of the ET_B receptor agonist BQ-3020 was completely blocked by 5 × 10⁶ M BQ-788 and partially inhibited by 5 × 10⁶ M of the phospholipase C (PLC) inhibitor 2-nitro-4-carboxyl-N,N-diphenylcarbamate (NCDC), an L-type Ca²⁺ channel antagonist (nifedipine), a protein kinase C (PKC) inhibitor (chelerythrine), or removal of Ca²⁺ from the perfusate (n = 4 or 5; P < 0.05). The vasoconstrictor effect of S6c was also partially blocked by 5 × 10⁶ M of NCDC, nifedipine, or chelerythrine or by removal of Ca²⁺ from the perfusate (n = 4; P < 0.01). We conclude that ET_B receptors play a central role in mediating ET-1-induced vasoconstriction in pig skin, and the mechanism probably involves L-type Ca²⁺ channels, PLC, and PKC.