Considerable attention has focused on the role of protein kinase C in triggering the profound infarct-sparing effect of ischemic preconditioning (PC). In contrast, the involvement of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃), the second messenger generated in parallel with the diacylglycerol-PKC pathway, remains poorly understood. We hypothesized that, if Ins(1,4,5)P₃ signaling (i.e., release of Ins(1,4,5)P₃ and subsequent binding to Ins(1,4,5)P₃ receptors) contributes to PC-induced cardioprotection, then the reduction of infarct size achieved with PC would be attenuated in mice that are deficient in Ins(1,4,5)P₃ receptor protein. To test this concept, hearts were harvested from: (1) B6C3Fe-a/a-Itpr-1^opt+/-/J mutants displaying reduced expression of Ins(1,4,5)P₃ receptor-1 protein; (2) Itpr-1^opt+/+ wild-types from the colony; and (3) C57BL/6J mice. All hearts were buffer-perfused and randomized to receive: 2 5-min episodes of PC ischemia; pretreatment with D-myo-Ins(1,4,5)P₃ (sodium salt of native Ins(1,4,5)P₃); the mitochondrial K_ATP channel opener diazoxide; or no intervention (controls). After the treatment phase, all hearts underwent 30 min global ischemia followed by 2 hours of reperfusion, and infarct size was delineated by tetrazolium staining. In both wild-type and C57BL/6J cohorts, area of necrosis in hearts that received PC, D-myo-Ins(1,4,5)P₃ and diazoxide averaged 28-35% of the total LV, significantly smaller than the values of 52-53% seen in controls (p<.05). In contrast, in Itpr-1^opt+/- mutants, protection was only seen with diazoxide: neither PC nor D-myo-Ins(1,4,5)P₃ limited infarct size (52-58% versus 56% of the LV in mutant-controls). These data provide novel evidence that Ins(1,4,5)P₃ signaling contributes to infarct size reduction with preconditioning.