Our recent study indicates that homocysteine (Hcy) plays a detrimental role in ischemia-reperfusion induced renal injury (Am. J. Physiology Renal Physiol 292:F1354-1363, 2007). Elevation of renal Hcy concentration during ischemia-reperfusion is attributed to reduced activity of cystathionine--synthase (CBS) that catalyzes the rate-limiting step in the transsulfuration pathway for metabolism of majority of Hcy in the kidney. However, the mechanisms of impaired CBS activity in the kidney are unknown. The aim of this study was to investigate the effects of pH and nitric oxide (NO) on the CBS activity in the kidney during ischemia-reperfusion. The left kidney of Sprague-Dawley rat was subjected to ischemia-reperfusion. The CBS activity was significantly reduced in kidneys subjected to ischemia alone (15 to 60 min) or subjected to ischemia followed by reperfusion for 1 to 24-h. The pH was markedly reduced in kidneys upon ischemia. Injection of alkaline solution into the kidney partially restored the CBS activity during ischemia. Further analysis revealed that reduction of CBS activity during reperfusion was accompanied by an elevation of NO metabolites (nitrate and nitrite) in the kidney tissue. Injection of a NO scavenger, 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), restored the CBS activity in kidneys subjected to ischemia-reperfusion. Treatment with PTIO could abolish ischemia-reperfusion induced lipid peroxidation and prevent cell death in the kidney. These results suggested that metabolic acidosis during ischemia and accumulation of NO metabolites during reperfusion contributed, in part, to reduced CBS activity leading to an elevation of renal Hcy levels, which in turn, played a detrimental role in the kidney.