Hepatic blood supply is uniquely regulated by the hepatic arterial buffer response (HABR), counteracting alterations of portal venous blood flow by flow changes of the hepatic artery. Hydrogen sulfide (H₂S) has been recognized as a novel signaling molecule with vasoactive properties. However, the contribution of H₂S in mediating the HABR is not studied yet. In pentobarbital-anesthetized and laparotomized rats, flow probes around the portal vein and hepatic artery allowed for assessment of the portal venous (PVBF) and hepatic arterial blood flow (HABF) under baseline conditions and stepwise reduction of PVBF for induction of HABR. Animals received either the H₂S donor Na₂S, DL-propargylglycine as inhibitor of the H₂S synthesizing enzyme cysthationine--lyase (CSE), or saline alone. Additionally, animals were treated with Na₂S and the K_ATP channel inhibitor glibenclamide or with glibenclamide alone. Na₂S markedly increased the buffer capacity to 27.4 ± 3.0% (P <0.05 vs controls: 15.5 ± 1.7%), while blockade of H₂S formation by DL-propargylglycine significantly reduced the buffer capacity (8.5 ± 1.4%). Glibenclamide completely reversed the H₂S-induced increase of buffer capacity to the control level. By means of RT-PCR, Western blot analysis and immunohistochemistry, we observed the expression of both H₂S synthesizing enzymes (CSE and cystathionine--synthase (CBS)) in aorta, V. cava, hepatic artery and portal vein as well as in hepatic parenchymal tissue. Terminal branches of the hepatic afferent vessels expressed only CSE. We show for the first time that CSE-derived H₂S contributes to HABR and partly mediates vasorelaxation of the hepatic artery via activation of K_ATP channels.