When applied to rat sciatic nerve, endothelin-1 (ET-1) induces acute pain behavior that is blocked by coadministration of an endothelin A (ET A ) receptor-selective antagonist. To characterize the pharmacology of this effect, we applied (1) different concentrations (40 to 800 gmmol/L) of ET-1 to the surface of sciatic nerve, (2) a second dose of ET-1, 1 to 24 hours after the initial dose, (3) an ET A receptor-selective antagonist (BQ-123) to nerve after induction of pain behavior by ET-1, or (4) ET-1 intraneurally to determine, respectively, the dose-dependence, pharmacokinetics, desensitization, reversibility, and likely tissue site of action for ET-1 in inducing pain behavior. In vitro and in vivo experiments also were performed to determine the degree of saturable binding of ET A and ET B receptors, and to detect correlations between this binding and pain behavior. Maximum hindpaw flinch frequency was observed at a concentration of 400 μmol/L ET-1, with estimated EC 5 0 of 250 to 300 μmol/L for events assayed at 10 and 15 minutes after drug application. Repeat application of 400 μmol 1 to 24 hours after initial application produced no flinching, suggesting persistent behavioral desensitization to the effects of ET-1. Complete desensitization to 400 μmol/L ET-1 also was observed 75 minutes after an initial application of 100 μmol/L ET-1, a concentration that produced minimal flinching, suggesting that desensitization occurred secondary to receptor occupancy. Extraneural administration of BQ-123 12 minutes after ET1-induced hindpaw flinching had begun reversed this pain behavior within 5 to 10 minutes, implying reversibility of ET-1 binding and the potential clinical usefulness of such agents. Intraneural ET-1 also induced flinching, but at one tenth the dose used in extraneural experiments and with an earlier peak effect, pointing to an intraneural site of action for extraneural ET-1. Although saturable (BQ-123-sensitive) binding was measured in vitro in 4 and 40 nmol/L ET-1 solutions (B m a x ~ 10 fmol/mg wet nerve), extraneural application of 400 μmol/L ET-1 in vivo resulted in too high an ET-1 content (1 to 4 pmol/mg wet nerve) for any saturable binding to be detected. These results establish that acute pain behavior induced by ET-1 applied to rat sciatic nerve is dose-dependent, subject to desensitization with repeat application, reversible, and likely caused by actions within the intraneural compartment.