Two protein signaling systems, phosphorylation and S-nitrosylation, influence most aspects of cellular physiology. S-nitrosylation, which generates a nitrosothiol linkage on cysteine residues, is caused by nitric oxide (NO). NO is believed to act as an anti-apoptotic agent by inhibiting caspase activity in cardiomyocytes, but there is little direct evidence for this. We investigated whether apoptosis inhibition by NO involved S-nitrosylation of caspases in doxorubicin (DOX)-induced myocardial apoptosis. Cardiomyocytes were treated with 1 micromol/l of DOX to induce apoptosis. Pretreatment with an NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) reduced the apoptosis. This effect was attenuated by treatment with 100 micromol/l of mercury dichloride (HgCl2), which is an agent of denitrosylation. After 24 h DOX-treatment, SNAP reduced the increased caspase-3 activity by 63%, and this effect was reversed by treatment with HgCl2. Immunoblot analysis showed that accumulation of the cleaved caspase-3 protein, an active form that induces apoptosis was inhibited significantly by SNAP. To elucidate nitrosothiol formation on caspase-3 by NO, we did several experiments. First, we prepared an immunoprecipitate of caspase-3 and measured the concentration of NO released from the precipitated complex by HgCl2. Second, S-nitrosylated proteins, purified by immunoprecipitation of caspase-3, were biotinylated and the biotin concentration was estimated by immunoblotting. Third, dual immunofluorescent staining was done with antibodies for S-nitrosocysteine and caspase-3. Results showed that formation of nitrosothiol in caspase-3 in DOX-treated cardiomyocytes with SNAP was increased significantly compared with untreated cardiomyocytes. We reported here that exogenous NO produces an anti-apoptotic effect by suppression of caspase activity via S-nitrosylation in cardiomyocytes.