The objective of this study is to assess the participation of mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels in the cardioprotective effects of the Na(+)/H(+) exchanger (NHE-1) blocker cariporide in isolated rat hearts. Regional ischemia was induced by occlusion of left anterior descending coronary artery during 40 min followed by 2-h reperfusion (IC). Cariporide (C, 10 μΜ), or C plus 5-hydroxydecanoate (5-HD, 100 μM, a selective mitoK(ATP) channel inhibitor), or C plus chelerythrine (Chele, 1 μM, a PKC inhibitor), or an opener of mitoK(ATP) channels, diazoxide (Dz, 100 μM) was applied at the onset of reperfusion. Infarct size (IS) and myocardial function were evaluated. The calcium-induced permeability transition pore (mPTP) opening was determined by measuring the light scattering decrease (LSD, a.u.) in isolated mitochondria in the absence and presence of C, C + 5-HD and Dz. IS was 33 ± 2% of the risk area in IC and was significantly diminished by C (15 ± 2%, p < 0.05), which also improved myocardial function [LVDP = 58 ± 5% (IC) vs 80 ± 5% (C)] and blunted LSD [0.80 ± 0.04 (IC) vs 0.51 ± 0.04 (C) a.u.]. 5-HD and Chele were both able to abolish the cardioprotective effects of C on IS. Dz treatment decreased IS and LSD to a similar extent to that produced by C (15 ± 4% and 0.52 ± 0.04 a.u., respectively). The present data suggest that attenuation of mPTP opening after PKC-mediated mitoK(ATP) channel activation is a crucial step for the cardioprotective effects of cariporide.