Inhibition of mitochondrial permeability transition pore (MPTP) opening at reperfusion is critical for cardioprotection by ischemic preconditioning (IP). Some studies have implicated mitochondrial protein phosphorylation in this effect. Here we confirm that mitochondria rapidly isolated from preischemic control and IP hearts show no significant difference in calcium-mediated MPTP opening, whereas IP inhibits MPTP opening in mitochondria isolated from IP hearts following 30 minutes of global normothermic ischemia or 3 minutes of reperfusion. Analysis of protein phosphorylation in density-gradient purified mitochondria was performed using both 2D and 1D electrophoresis, with detection of phosphoproteins using Pro-Q Diamond or phospho-amino-specific antibodies. Several phosphoproteins were detected, including voltage-dependent anion channels isoforms 1 and 2, but none showed significant IP-mediated changes either before ischemia or during ischemia and reperfusion, and neither Western blotting nor 2D fluorescence difference gel electrophoresis detected translocation of protein kinase C (, , or isoforms), glycogen synthase kinase 3, or Akt to the mitochondria following IP. In freeze-clamped hearts, changes in phosphorylation of GSK3, Akt, and AMP-activated protein kinase were detected following ischemia and reperfusion but no IP-mediated changes correlated with MPTP inhibition or cardioprotection. However, measurement of mitochondrial protein carbonylation, a surrogate marker for oxidative stress, suggested that a reduction in mitochondrial oxidative stress at the end of ischemia and during reperfusion may account for IP-mediated inhibition of MPTP. The signaling pathways mediating this effect and maintaining it during reperfusion are discussed.