Rat neonatal ventricular myocytes exposed to simulated ischaemia and reperfusion (SI/R) were used as an in vitro model to delineate the role(s) of extracellular signal–regulated kinase (ERK), p38 and c–Jun NH₂–terminal protein kinase (JNK), as well as PKB in apoptosis. Exposure of the myocytes to SI (simulated ischaemia – energy depletion induced by KCN and 2-deoxy-D–glucose) reduced cell viability, as measured by the 3–[4,5–dimethylthiazol–2–yl]–2,5–diphenyl tetrazolium bromide (MTT) assay, and stimulated apoptosis as evidenced by caspase–3 activation and poly(ADP–ribose) polymerase (PARP) cleavage. However, morphological evidence of increased apoptosis, detected by staining with Hoechst 33342, was only seen in response to reperfusion. This suggests that although ischaemic conditions are sufficient to induce cellular markers of apoptosis (PARP cleavage and caspase–3 activation), reperfusion is required to complete the apoptotic pathway in these cells. Furthermore, SI resulted in a rapid, strong, biphasic activation of p38 concomitant with a weak and transient activation of the two ERK isoenzymes, p42/p44–MAPK. Reperfusion for 5 minutes resulted in a strong phosphorylation of p42/p44–MAPK, while no additional p38 activation was seen at this stage. On the other hand, p46/p54–MAPK (JNK) was phosphorylated in response to 5 minutes of reperfusion only and not during SI alone. A peak of PKB/Akt (Ser⁴⁷³) activity was seen within 5 minutes of exposure to SI, whereas PKB/Akt (Thr³⁰⁸) phosphorylation remained at the baseline level. Both PKB/Akt phosphorylation sites (Ser⁴⁷³ and Thr³⁰⁸) were phosphorylated after 5 minutes of reperfusion. Inhibition of PI–3–kinase activity, using wortmannin, decreased phosphorylation on both sites during SI. However, only SI/R-induced PKB/Akt phosphorylation on Thr³⁰⁸ was reduced by wortmannin. Myocytes pre–treated with SB203580, a p38–inhibitor, displayed a significant increase in cell viability [63.67 ± 1.85 to 84.33 ± 4.8% (p < 0.05)] and attenuation of the apoptotic index during SI/R [22.6 ± 2.94% to 9 ± 0.43% (p < 0.001)], while SP600125, a specific JNK inhibitor, caused a significant increase in caspase–3 activation [1.66 ± 0.03 fold to 2.56 ± 0.27 fold (p < 0.001)] and apoptotic index [22.6 ± 2.94% to 32.75 ± 6.13% (p < 0.05)]. However, PD98059, an ERK inhibitor, failed to affect apoptosis during SI/R. Inhibition of PI–3–kinase prevented the increase in mitochondrial viability usually observed during reperfusion. Interestingly, wortmannin caused a significant increase in PARP cleavage during reperfusion, but had no effect on caspase–3 activation or the apoptotic index. Our results suggest that p38 has a pro–apoptotic role while JNK phosphorylation is protective in our cell model and that these kinases act via caspase–3 to prevent or promote cell survival in response to SI/R–induced injury.