The possibility of a direct mitochondrial action of Na⁺/H⁺ exchanger-1 (NHE-1) inhibitors decreasing reactive oxygen species (ROS) production was assess in cat myocardium. Angiotensin II and endothelin-1 induced an NADPH oxidase-dependent (NOX) increase in anion superoxide (O₂^-) production detected by chemiluminescence. Three different NHE-1 inhibitors-cariporide, BIIB723, and EMD87580-with no ROS scavenger activity prevented this increase. The mitochondria appeared to be the source of the NOX-dependent ROS released by the "ROS-induced ROS release mechanism" that was blunted by the mitochondrial K_ATP channel (mK_ATP) blockers 5-hydroxydecanoate and glibenclamide, inhibition of complex I of the electron transport chain with rotenone and inhibition of the permeability transition pore (MPTP) by cyclosporin A. Cariporide also prevented O₂^- production induced by the opening of mK_ATP with diazoxide. Ca²⁺-induced swelling was evaluated in isolated mitochondria as an indicator of MPTP formation. Cariporide decreased mitochondrial swelling to the same extent as cyclosporin A and bongkrekic acid confirming its direct mitochondrial action. Increased O₂^- production, as expected, stimulated ERK1/2 and p90^RSK phosphorylation. This was also prevented by cariporide giving additional support to the existence of a direct mitochondrial action of NHE-1 inhibitors in preventing ROS release. In conclusion, we report a mitochondrial action of NHE-1 inhibitors that should lead us to revisit or reinterpret previous landmark observations about their beneficial effect in several cardiac diseases, such as ischemia/reperfusion injury and cardiac hypertrophy and failure. Further studies are needed to clarify the precise mechanism and site of action of these drugs in blunting MPTP formation and ROS release.