Hyperhomocysteinemia is widely recognized as an independent risk factor for coronary artery vascular disease, although the underlying mechanisms are not well understood. This study aims to investigate the effect of homocysteine on nitric oxide ( NO) production in coronary microvascular endothelial cells (CMECs) and putative mechanisms mediating this effect. CMECs were isolated on Langendorff system by collagenase perfusion of hearts from male rats and cultured. The effect of homocysteine (0.01 to 1 mM) on basal and stimulated NO production was evaluated by measuring nitrite in the culture media after incubation with or without N-G-nitro-L-arginine methyl ester (L-NAME) ( 1 mM), superoxide dismutase (100 U/mL), or catalase ( 1000 U/mL) for 24 h. Total nitrite was measured using Griess reaction after reduction of nitrate to nitrite with nitrate reductase. Homocysteine did not affect basal nitrite accumulation; however, it significantly increased the nitrite accumulation induced by the calcium ionophore A23187 or interleukin-1 beta only at 1 mM. This effect of homocysteine was significantly inhibited by L-NAME, superoxide dismutase, and catalase. In conclusion, homocysteine increases NO release from stimulated CMECs without affecting basal NO production, which is probably accompanied by increased production of reactive oxygen species. It can be postulated that endothelial cells generate NO in order to minimize the damage caused by homocysteine.