Objectives: Recruitment of the survival kinase cascades, PI3K–Akt and Raf–MEK1/2–Erk1/2, at the time of reperfusion, following a lethal ischemic insult, may mediate the protection associated with ischemic preconditioning (IPC). The exact interplay between these two kinase cascades in mediating this effect is not clear. We examine the ‘cross-talk’ between these kinase cascades in their contribution to IPC-induced protection. Methods and results: In isolated perfused rat hearts subjected to 35 min of lethal ischemia±ischemic preconditioning, the phosphorylation states of Akt, Erk1/2, p70S6K were determined after 15 min of reperfusion, and infarct size was measured after 120 min of reperfusion. IPC induced a threefold increase in Akt, Erk1/2, and p70S6K phosphorylation, at reperfusion. We found that inhibiting the PI3K–Akt (using LY294008) at reperfusion induced the phosphorylation of Erk1/2–p70S6K, and conversely, that inhibiting the MEK1/2–Erk1/2 pathway (using PD 98059) at reperfusion, induced the phosphorylation of Akt, suggesting ‘cross-talk’ between the two kinase pathways. However, this effect was not accompanied by a reduction in infarct size (43.1±7.2% with LY 294008 and 57.7±7.0% with PD 98059 vs. 46.3±5.8% in control; P=NS), suggesting that both the kinase cascades may need to be activated to mediate IPC-induced protection. IPC reduced the infarct–risk volume ratio to 17.8±2.3% from 46.3±5.8% in control (PPConclusions: We report that, the survival kinase cascades PI3K–Akt and MEK1/2–Erk1/2, which are recruited at the time of reperfusion in response to ischemic preconditioning, exhibit ‘cross-talk’ such that inhibiting one cascade activates the other and vice versa. Furthermore, at the time of reperfusion, these kinase cascades mediate IPC-induced protection, by acting in concert via p70S6K.