--- - |2+ Abstract Aims The epithelial sodium channel (ENaC) is expressed in cultured endothelial cells and inhibitory coupling to eNOS activity has been proposed. The present study tested the hypothesis that ENaC blockers increase systemic NO‐products and lower blood pressure in patients and mice, depending on eNOS. Methods NO‐products and cGMP were measured in diabetes patient urine and plasma samples before and after amiloride treatment (20‐40 mg for two days, plasma n = 22, urine n = 12 and 5‐10 mg for eight weeks, plasma n = 52, urine n = 55). Indwelling catheters were implanted in the femoral artery and vein in mice for continuous arterial blood pressure and heart rate recordings and infusion. Results Treatment with amiloride for two days increased plasma and urine NO‐products, while plasma cGMP decreased and urinary cGMP was unchanged in patient samples. Eight weeks of treatment with amiloride did not alter NO‐products and cGMP. In mice, amiloride boli of 5, 50, and 500 µg/kg lowered heart rate and arterial blood pressure significantly and acutely. Benzamil had no effect on pressure and raised heart rate. In hypertensive eNOS−/− and L‐NAME‐treated mice, amiloride lowered blood pressure significantly. L‐NAME infusion significantly decreased NO‐products in plasma; amiloride and eNOS‐deletion had no effect. An acetylcholine bolus resulted in acute blood pressure drop that was attenuated in eNOS−/− and L‐NAME mice. ENaC subunit expressions were not detected consistently in human and mouse arteries and endothelial cells. Conclusion Amiloride has an acute hypotensive action not dependent on ENaC and eNOS and likely related to the heart. - 'Acta Physiologica, EarlyView. '