Pretreatment with tumor necrosis factor-alpha (TNF-alpha) is known to trigger cardioprotection. TNF-alpha can activate multiple downstream signaling cascades. However, it is not known whether the mitochondrial permeability transition pore (MitoPTP) is involved in TNF-alpha-induced cardioprotection. In the present study, we examined whether TNF-alpha inhibits MitoPTP opening. In isolated rat hearts subjected to 30 min regional ischemia and 120 min reperfusion, pretreatment with 10 U/ml TNF-alpha for 7 min followed by 10 min washout improved the recovery of left ventricular developed pressure (LVDP) and rate-pressure product (RPP = LVDP x heart rate) during reperfusion and reduced the infarct size. Administration of 20 micromol/L atractyloside, a MitoPTP opener, for 20 min (last 5 min of ischemia and first 15 min of reperfusion) and pretreatment with 1 mu inhibitor of the Ca2+-activated K+ mol/L paxilline, an channel, for 5 min before ischemia, attenuated the recovery of LVDP and RPP and the reduction of infarct size induced by TNF-alpha. The findings indicate that, in the isolated heart model, TNF-alpha protects myocardium against ischemia/reperfusion injury via inhibiting MitoPTP opening as well as by activating the Ca2+-activated K+ channel.