Endothelin-1 (ET-1) inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (TALH) expresses ET-1 receptors. In many tissues, activation of ET(B) receptors stimulates release of NO, and we recently reported that endogenous NO inhibits TALH chloride flux (J(Cl)). However, the relationship between ET-1 and NO in the control of nephron transport has not been extensively studied. We hypothesized that ET-1 decreases NaCl transport by cortical TALHs through activation of ET(B) receptors and release of NO. Exogenous ET-1 (1 nM) decreased J(Cl) from 118.3 ± 15.0 to 62.7 ± 13.6 pmol · mm-1 · min-1 (48.3 ± 8.2% reduction), whereas removal of ET-1 increased J(Cl) in a separate group of tubules from 87.6 ± 10.7 to 115.2 ± 10.3 pmol · mm-1 · min-1 (34.5 ± 6.2% increase). To determine whether NO mediates the inhibitory effects of ET-1 on J(Cl), we examined the effect of inhibiting of NO synthase (NOS) with N(G)-nitro-L-arginine methyl ester (L-NAME) on ET-1-induced changes in J(Cl). L-NAME (5 mM) completely prevented the ET-1-induced reduction in J(Cl), whereas D-NAME did not. L-NAME alone had no effect on J(Cl). These data suggest that the effects of ET-1 are mediated by NO. Blockade of ET(B) receptors with BQ-788 prevented the inhibitory effects of I nM ET-1. Activation of ET(B) receptors with sarafotoxin S6c mimicked the inhibitory effect of ET-1 on J(Cl) (from 120.7 ± 12.6 to 75.4 ± 13.3 pmol · mm-1 · min-1). In contrast, ET(A) receptor antagonism with BQ-610 did not prevent ET-1-mediated inhibition of TALH J(Cl) (from 96.5 ± 10.4 to 69.5 ± 8.6 pmol · mm-1 · min-1). Endothelin increased intracellular calcium from 96.9 ± 14.0 to 191.4 ± 11.9 nM, an increase of 110.8 ± 26.1%. We conclude that exogenous endothelin indirectly decreases TALH J(Cl) by activating ET(B) receptors, increasing intracellular calcium concentration, and stimulating NO release. These data suggest that endothelin acts as a physiological regulator of TALH NO synthesis, thus inhibiting chloride transport and contributing to the natriuretic effects of ET-1 observed in vivo.