Renal endothelin-1 (ET-1) and purinergic signaling systems regulate Na+ reabsorption in the renal medulla. A link between the renal (ET-1) and purinergic systems was demonstrated in-vitro, however, the in-vivo interaction between these systems has not been defined. To test whether renal medullary activation of purinergic (P2) receptors promotes ET-dependent natriuresis, we determined the effect of increased medullary NaCl loading on Na+ excretion and inner medullary ET-1 mRNA expression in anesthetized adult male Sprague Dawley rats in the presence and absence of purinergic receptor antagonism. Isosmotic saline (NaCl; 284 mOsmol/kg H2O) was infused into the medullary interstitium (500 μl/h) during a 30 min baseline urine collection period, followed by isosmotic or hyperosmotic saline (1800 mOsmol/kg H2O) for two further 30 min urine collection periods. Na+ excretion was significantly increased during intramedullary infusion of hyperosmotic saline. Compared with isosmotic saline, hyperosmotic saline infused into the renal medulla caused significant increases in inner medullary ET-1 mRNA expression. Renal intramedullary infusion of the purinergic (P2) receptor antagonist, suramin, inhibited the increase in Na+ excretion and inner medullary ET-1 mRNA expression induced by NaCl loading in the renal medulla. Activation of medullary purinergic (P2Y2/4) receptors by infusion of UTP increased urinary Na+ excretion. Combined ETA and ETB receptor blockade abolished the natriuretic response to intramedullary infusion of UTP. These data demonstrate that activation of medullary purinergic (P2) receptors promotes ET-dependent natriuresis in male rats, suggesting that the renal ET-1 and purinergic signaling systems interact to efficiently facilitate excretion of a NaCl load.