Mitochondria generate reactive oxygen species (ROS) dependent on substrate conditions, O₂ concentration, redox state, and activity of the mitochondrial complexes. It is well known that the FADH₂-linked substrate succinate induces reverse electron flow to complex I of the electron transport chain and that this process generates superoxide (O₂^.-); these effects are blocked by the complex I blocker rotenone. We demonstrated recently that succinate + rotenone dependent H₂O₂ generation in isolated mitochondria increased mildly on activation of the putative mitochondrial Ca²⁺sensitive K⁺ channel (mtK_Ca) by low concentrations of NS-1619. In the present study we examined the effects of NS -1619 on mitochondrial O₂ consumption, membrane potential (m), and H₂O₂ release rates and redox state in guinea pig heart isolated mitochondria respiring on succinate but without rotenone. NS-1619 (30 µM) increased state 2 and state 4 respiration by 26±4% and 14±4%, respectively; this increase was abolished by the mtKCa channel blocker paxilline (5 µM). Paxilline alone had no effect on respiration. NS-1619 did not alter m or redox state but decreased H₂O₂ production by 73% vs. control; this was partially inhibited by paxilline. We conclude that under substrate conditions that allow reverse electron flow, matrix K⁺ influx through mtK_Ca channels reduces mitochondrial H₂O₂ production by accelerating forward electron flow. Our prior study showed that NS-1619 induced an increase in H₂O₂ generation with blocked reverse electron flow. These results support our prior study, which suggests that NS-1619 induced matrix K⁺ influx increases forward electron flow despite the high reverse electron flow, and emphasizes the importance of substrate conditions on interpretation of effects on mitochondrial bioenergetics.