Consensus has formed that hydrogen sulfide (H 2 S) has robust cytoprotective actions in multiple organ systems. The pro-survival actions and biological profiles of H 2 S are remarkably similar to those of nitric oxide (NO). Although there is recent evidence of cross-talk between H 2 S and NO, these molecules are thought to modulate independent signaling pathways.Mice devoid of the key H 2 S producing enzyme, cystathionine gamma-lyase (CSE KO), exhibited impaired endothelial nitric oxide synthase (eNOS) function and diminished circulating nitrite (0.41 uM vs 0.73μM, p<0.05) nitrosylated protein (3.37nM vs 15.03nM, p<0.05), and cyclic GMP (9.3 vs 28.05pmol/ml, p<0.01) levels compared to wild-type (WT) mice. Further studies revealed that H 2 S therapy in CSE KO mice restored eNOS function and NO levels. eNOS KO mice and mice with phospho-dead eNOS (S1179A) were subjected to 45min of ischemia, followed by 24h of reperfusion. The H 2 S donor, Na 2 S (100μg/kg), or vehicle (0.9% NaCl) was administered 5min before reperfusion. There was no significant change in myocardial infarction in the H 2 S-treated group compared to the vehicle-treated group.These findings reveal that H 2 S modulates eNOS activity and nitric oxide signaling. Moreover, the inability of H 2 S therapy to protect eNOS dysfunctional mice against myocardial ischemia/reperfusion injury indicates that the cardioprotective actions of H 2 S are dependent on eNOS function and NO signaling.