Activation of protein kinase Cε (PKCε), opening of mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP), and increased mitochondrial reactive oxygen species (ROS) are key events in the signaling that underlies cardioprotection. We showed previously that mitoK_ATP is opened by activation of a mitochondrial PKCε, designated PKCε1, that is closely associated with mitoK_ATP. mitoK_ATP opening then causes an increase in ROS production by complex I of the respiratory chain. This ROS activates a second pool of PKCε, designated PKCε2, which inhibits the mitochondrial permeability transition (MPT). In the present study, we measured mitoK_ATP-dependent changes in mitochondrial matrix volume to further investigate the relationships among PKCε, mitoK_ATP, ROS, and MPT. We present evidence that 1) mitoK_ATP can be opened by H₂O₂ and nitric oxide (NO) and that these effects are mediated by PKCε1 and not by direct actions on mitoK_ATP, 2) superoxide has no effect on mitoK_ATP opening, 3) exogenous H₂O₂ or NO also inhibits MPT opening, and both compounds do so independently of mitoK_ATP activity via activation of PKCε2, 4) mitoK_ATP opening induced by PKG, phorbol ester, or diazoxide is not mediated by ROS, and 5) mitoK_ATP-generated ROS activates PKCε1 and induces phosphorylation-dependent mitoK_ATP opening in vitro and in vivo. Thus mitoK_ATP-dependent mitoK_ATP opening constitutes a positive feedback loop capable of maintaining the channel open after the stimulus is no longer present. This feedback pathway may be responsible for the lasting protective effect of preconditioning, colloquially known as the memory effect.