Hydrogen sulfide (H₂S), a novel endogenous gaseous bioactive substance, has recently been implicated in the regulation of cardiovascular and neuronal functions. However, its role in the control of renal function is unknown. In the present study, incubation of renal tissue homogenates with l-cysteine (l-Cys) (as a substrate) produced H₂S in a concentration-dependent manner. This H₂S production was completely abolished by inhibition of both cystathionine β-synthetase (CBS) and cystathionine γ-lyase (CGL), two major enzymes for the production of H₂S, using amino-oxyacetic acid (AOAA), an inhibitor of CBS, and propargylglycine (PPG), an inhibitor of CGL. However, inhibition of CBS or CGL alone induced a small decrease in H₂S production. In anesthetized Sprague-Dawley rats, intrarenal arterial infusion of an H₂S donor (NaHS) increased renal blood flow, glomerular filtration rate (GFR), urinary sodium (U_Na·V), and potassium (U_K·V) excretion. Consistently, infusion of both AOAA and PPG to inhibit the endogenous H₂S production decreased GFR, U_Na·V, and U_K·V, and either one of these inhibitors alone had no significant effect on renal functions. Infusion of l-Cys into renal artery to increase the endogenous H₂S production also increased GFR, U_Na·V, and U_K·V, which was blocked by AOAA plus PPG. It was shown that H₂S had both vascular and tubular effects and that the tubular effect of H₂S might be through inhibition of Na⁺/K⁺/2Cl^- cotransporter and Na⁺/K⁺/ATPase activity. These results suggest that H₂S participates in the control of renal function and increases urinary sodium excretion via both vascular and tubular actions in the kidney.