We previously reported that activation of phosphatidylinositol-3-kinase (PI3-kinase) is involved in ischemic preconditioning (PC). Our goal was to determine downstream targets of PI3-kinase. In perfused rat hearts, PC (4 cycles of 5 minutes of ischemia and 5 minutes of reflow) increased phosphorylation of glycogen synthase kinase-3&bgr; (GSK-3&bgr;), a downstream target of PI3-kinase and protein kinase B (PKB), an effect that was blocked by wortmannin. Because phosphorylation inactivates GSK-3&bgr;, we examined whether PC-induced phosphorylation and inhibition of GSK-3&bgr; is important in PC by using two inhibitors of GSK-3&bgr;, lithium and SB 216763. Pretreatment of perfused rat hearts with lithium or SB 216763, before ischemia, mimicked the protective effects of PC; hearts treated with either lithium or SB 216763 had improved postischemic function and reduced infarct size. These findings indicate that inhibition of GSK-3&bgr; is protective and that this PI3-kinase–dependent signaling pathway may play an important role in ischemic preconditioning.