The present study was conducted to examine the role of a major cardiac phospholipase C (PLC) isozyme, PLC ₁, in cardiomyocytes during oxidative stress. Left ventricular cardiomyocytes were isolated by collagenase digestion from adult male Sprague-Dawley rats (250-300 g) and treated with 20, 50 and 100 µM hydrogen peroxide (H₂O₂) for 15 min. A concentration-dependent increase in the mRNA level and membrane protein content of PLC ₁ was observed with H₂O₂ treatment. Furthermore, PLC ₁ was activated in response to H₂O₂, as revealed by an increase in the phosphorylation of its tyrosine residues. There was a marked increase in the phosphorylation of the anti-apoptotic protein, Bcl-2 by H₂O₂; this change was attenuated by a PLC inhibitor, U73122. Although both protein kinase C (PKC) and protein contents were increased in the cardiomyocyte membrane fraction in response to H₂O₂, PKC activation, unlike PKC was attenuated by U73122 (2 µM). Inhibition of PKC with inhibitory peptide (0.1 µM) prevented Bcl-2 phosphorylation. Moreover different concentrations (0.05, 0.1 and 0.2 µM) of this peptide augmented the decrease in cardiomyocyte viability in response to H₂O₂. In addition, a decrease in cardiomyocyte viability, as assessed by trypan blue exclusion, due to H₂O₂ was also seen when cells were pretreated with U73122 and was as a result of increased apoptosis. It is therefore suggested that PLC ₁ may play a role in cardiomyocyte survival during oxidative stress via PKC and phosphorylation of Bcl-2.