Post-ischemic interventions that activate phosphatidylinositol-3-OH kinase (PI3K)-Akt or ERK1/2 pro-survival kinases (the so-called \"reperfusion injury signalling kinase (RISK) pathway\") during the first few minutes of reperfusion protect against lethal reperfusion-induced injury. We have previously shown that insulin protects against reperfusion-induced injury via activation of the PI3K-Akt pathway. In addition, opening of the mitochondrial permeability transition pore (mPTP) at the time of reperfusion is a major determinant of lethal reperfusion-induced injury, and pharmacologically inhibiting it is cardioprotective. In this study, we examined the relationship between the pro-survival kinase pathways and mPTP opening. Specifically, we tested the hypothesis that activation of the pro-survival kinase pathway by insulin protects cardiomyocytes by reducing the probability of mPTP opening upon reperfusion. Laser illumination of the fluorophore, tetramethyl rhodamine methyl ester (TMRM), was used to induce oxidative stress in the preparation of adult rat ventricular cardiomyocytes. Maintained illumination ultimately induces mPTP opening, detected as a global mitochondrial depolarization, followed by ATP depletion and rigor contracture. Insulin significantly delayed mPTP opening by a factor of approximately 1.7-fold (P<0.001). The effect of insulin was prevented by Wortmannin and by LY-294002, inhibitors of the PI3K pathway, by SH-6, a selective inhibitor of Akt, and by L-NAME, an inhibitor of nitric oxide production. The expression of a dominant negative construct of Akt eliminated the effect of insulin in delaying mPTP opening in a cardiac cell line. Furthermore, the overexpression of constitutively active Akt was sufficient to maximally delay mPTP opening. These results indicate that activation of the PI3K-Akt pro-survival kinase pathway inhibits opening of the mPTP, and demonstrate an important link between the survival kinases and the mPTP