BACKGROUND/AIM: This study was designed to provide further evidence for the interactions between hydrogen sulfide (HS) and nitric oxide (NO) in ischemia/reperfusion (I/R) injury. MATERIALS AND METHODS: Rat hearts were studied with the Langendorff technique using the HS donor sodium hydrosulfide (NaHS, 40 μM) and the cystathionine gamma-lyase (CTH or CSE) inhibitor DL-propargylglycine (PAG, 1 mM). NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME, 30 mg/kg, 7 days) was administered before the isolation. The hearts were homogenized for biochemical and molecular analysis. RESULTS: NaHS reversed I/R-induced cardiac performance impairment, increased tissue nitric oxide production and decreased tissue markers for cardiac injury, while L-NAME inhibited these effects. The expression of CTH was increased with PAG, which was suppressed by L-NAME. CONCLUSION: HS and NO increase each other's production suggesting their interaction and cooperation in cardioprotection against I/R injury.