Objective: This study was designed to investigate the cardioprotective effect of the novel lipophilic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) against the oxidative stress exerted by H2O2 through the production of ⋅OH radical via the Fenton reaction and to compare them with those of the hydrophilic iron chelator deferoxamine (DFO) and the Na+/H+ exchange inhibitor methylisobutyl amiloride (MIA). Methods: We used long-term cultures of spontaneously beating adult guinea-pig ventricular cardiomyocytes developed and characterized previously in our laboratory. We assessed their contractile activity by video-recording as well as the underlying Cai2+ transients by Fura 2 fluorescence. In some experiments we also recorded these functional parameters, plus the electrical activity (action potentials) in response to electrical stimulation via suction pipettes, in individual freshly isolated myocytes. Results: Exposure of the regularly and synchronously beating cultured cardiomyocytes to 100 μM H2O2 initially caused a substantial prolongation of Cai2+ transients accompanied by an irregular contractile activity, then in contractile arrest and a severalfold increase in cytosolic [Ca2+] that occurred, within 30 min of H2O2 application. Similar effects were also observed using freshly isolated cardiomyocytes. The latter effects were first accompanied by significant prolongation of the action potential duration (APD) with superimposed early afterdepolarizations followed by a second phase with a very fast decrease in APD, contractions, as well as Cai2+ transients and a third phase of inexcitability, contractile arrest, increased cytoplasmic [Ca2+] and a final contracture. All these effects were irreversible in both types of preparations but they could be fully prevented by a 15-min preincubation with 200 μM SIH. Similar protective effects were observed with DFO, but in this case a much higher concentration had to be used (1 mM) and much longer (2 h) preincubation was needed. By contrast, 5 μM MIA failed to fully protect the cardiomyocytes, although a significant delay (10 min) of the effects of H2O2 was observed. Conclusions: The data indicate that SIH provides a very powerful and very fast protection against the oxidative stress exerted by H2O2 presumably via the iron-mediated Fenton reaction producing hydroxyl radical (⋅OH), whereas the protective effect of DFO is hindred by its very slow and rather limited intracellular entry, and the protection that MIA exerts via the inhibition of Na+/H+ exchange against H2O2 much less effective.