Prophylactic treatment with D-myo-inositol 1,4,5-trisphosphate hexasodium (D-myo-IP3), the sodium salt of the endogenous second messenger IP3, triggers a reduction of infarct size comparable in magnitude to that seen with ischemic preconditioning (PC). The mechanisms underlying D-myo-IP3-induced protection are, however, unknown. Accordingly, our aim was to interrogate the role of four archetypal mediators implicated in PC and other cardioprotective strategies - i.e., PKC, PI3-kinase/Akt, mitochondrial and/or sarcolemmal K_ATP channels - in the infarct-sparing effect of D-myo-IP3. Fifteen groups of isolated buffer-perfused rabbit hearts (five treated with D-myo-IP3, five that received PC, and five control cohorts) underwent 30 min of coronary artery occlusion and 2 h of reflow. One set of control, D-myo-IP3 and PC groups received no additional treatment, while the remaining sets were infused with chelerythrine, LY 294002, 5-HD, or HMR 1098 (inhibitors of PKC, PI3-kinase, mitochondrial and sarcolemmal K_ATP channels, respectively). Infarct size (delineated by tetrazolium staining) was, as expected, significantly reduced in both D-myo-IP3- and PC-treated hearts versus controls. D-myo-IP3-induced cardioprotection was blocked by 5-HD but not HMR, thereby implicating the involvement of mitochondrial (but not sarcolemmal) KATP channels. Moreover, the benefits of D-myo-IP3 were abrogated by LY 294002 while, in contrast, chelerythrine no effect. These latter pharmacologic data were corroborated by immunoblotting: D-myo-IP3 evoked a significant increase in expression of phospho-Akt, but had no effect on the activation/translocation of the cardioprotective -isoform of PKC. Thus, PI3-kinase/Akt signaling and mitochondrial K_ATP channels participate in the reduction of infarct size afforded by prophylactic administration of D-myo-IP3.