Neonatal rat cardiomyocytes were cultured and cytotoxicity in cultured cardiomyocytes was induced by H₂O₂ over a wide concentration range (0.05～50 mmol/L) to assess dynamically the effect of H₂O₂ on cardiomyocytes. The results showed that application of ＜0.1 mmol/L H₂O₂ to cardiomyocytes caused accumulation of lipid peroxide(MDA) at 24 h, and phase changes of cellular proliferation cycle at 2h, which represented early biochemical changes. Exposure of cardiomyocytes to the increasing concentrations of H₂O₂ (1～5 mmol/L) induced progressively biochemical injury; the levels of LDH and MDA were significantly higher in the cardiomyocytes exposed to H₂O₂ than in control, with concomitant morphologic changes. When exposed to high concentrations (＞10 mmol/L), a large number of cardiomyocytes were found dead by MTT assay, and morphologic examination by HE-staining showed that cardiomyocytes contracted extremely, forming a large dropping areas of cardiomyocytes, concurrently with the marked increase of membrane permeability which caused LDH substantial leakage from myocytes to H₂O₂. It is proposed that H₂O₂ accumulation can induce cardiomyocyte cytotoxicity in a dose-and time-dependent manner. Treatment with low concentrations of H₂O₂ causes cardiomyocytes early slight biochemical changes which represent pre-apototic injurious features. High concentrations of H₂O₂ can progressively induce lipid peroxidation, which cause the severe damage of the cell membrane. With exposure of cardiomyocytes to H₂O₂, the magnitude of the cytotoxicity is modulated by horseradish peroxidase (HRP). It is suggested that HRP may protect cardiomyocytes against reactive oxygen species.