Cystathionine--synthase (CBS) catalyzes the rate-limiting step in the transsulfuration pathway for the metabolism of homocysteine (Hcy) in the kidney. Our recent study demonstrates that ischemia-reperfusion reduces the activity of CBS leading to Hcy accumulation in the kidney, which in turn contributes to renal injury. The CBS is also capable of catalyzing the reaction of cysteine with Hcy to produce hydrogen sulfide (H₂S), a gaseous molecule that plays an important role in many physiological and pathological processes. The aim of the present study was to examine the effect of ischemia-reperfusion on CBS-mediated H₂S production in the kidney and to determine whether changes in the endogenous H₂S generation had any impact on renal ischemia-reperfusion injury. The left kidney of Sprague-Dawley rat was subjected to 45 min ischemia followed by 6h reperfusion. The ischemia-reperfusion caused lipid peroxidation and cell death in the kidney. The CBS-mediated H₂S production was decreased, leading to a significant reduction in the renal H₂S level. The activity of cystathionine-gama-lyase (CGL), another enzyme responsible for endogenous H₂S generation, was not altered in the kidney upon ischemia-reperfusion. Partial restoration of CBS activity by intraperitoneal injection of the NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) not only increased renal H₂S levels but also alleviated ischemia-reperfusion induced lipid peroxidation and reduced cell damage in the kidney tissue. Furthermore, administration of an exogenous H₂S donor, NaHS (100 µg/kg), improved renal function. Taken together, these results suggest that maintenance of tissue H₂S level may offer a renal protective effect against ischemia-reperfusion injury.