Endothelin-1 (ET-1) plays an important role in the regulation of salt and water excretion in the kidney. Considerable in vitro evidence suggests that the renal medullary ETB receptor mediates ET-1-induced inhibition of electrolyte reabsorption by stimulating nitric oxide (NO) production. The present study was conducted to test the hypothesis that NO synthase 1 (NOS1) and protein kinase G (PKG) mediate the diuretic and natriuretic effects of ETB receptor stimulation in vivo. Infusion of the ETB receptor agonist, sarafotoxin S6c (S6c: 0.45 µg/kg/h), into the renal medulla of anesthetized, male Sprague-Dawley rats markedly increased the urine flow (UV) and urinary sodium excretion (UNaV) by 67 and 120%, respectively. This was associated with an increase in medullary cGMP content, but did not affect blood pressure. In addition, S6c-induced diuretic and natriuretic responses were absent in ETB receptor-deficient rats. Co-infusion of NG-propyl-L-arginine (10 µg/kg/h), a selective NOS1 inhibitor suppressed S6c-induced increases in UV, UNaV and medullary cGMP concentrations. Rp-8-Br-PET-cGMPS (10 µg/kg/h) or DT-3 (18 µg/kg/h), a PKG inhibitor, also inhibited S6c-induced increases in UV and UNaV. These results demonstrate that renal medullary ETB receptor activation induces diuretic and natriuretic responses through a NOS1, cGMP and PKG pathway.