Recent evidence suggests that opening of mitochondrial K(ATP) channels in cardiac muscle triggers the preconditioning phenomenon through free radical production. The present study tested the effects of K(ATP) channel openers in a vascular smooth muscle cell model using the fluorescent probe MitoTracker (MTR) Red trade mark for detection of reactive oxygen species (ROS). Rat aortic smooth muscle cells (A7r5) were incubated with 1 micro M reduced MTR (non-fluorescent) and the MTR oxidation product (fluorescent) was quantified. Thirty-minute pretreatment with either diazoxide (200 micro M) or pinacidil (100 micro M), both potent mitochondrial K(ATP) channel openers, increased fluorescent intensity (FI) to 149 and 162 % of control (p < 0.05 for both), respectively, and the K(ATP) channel inhibitor 5-hydroxydecanoate (5 HD) blocked it. Valinomycin, a potassium-selective ionophore, raised FI to 156 % of control (p <: 0.05). However, 5 HD did not affect the valinomycin-induced increase in FI. Inhibition of mitochondrial electron transport (myxothiazol) or uncoupling of oxidative phosphorylation (dinitrophenol) also blocked either valinomycin- or diazoxide-induced increase in FI, and free radical scavengers prevented any diazoxide-mediated increase in fluorescence. Finally the diazoxide-induced increase in fluorescence was not blocked by the PKC inhibitor chelerythrine, but was by HMR 1883, a putative surface K(ATP) channel blocker. Thus opening of K(ATP) channels increases generation of ROS via the mitochondrial electron transport chain in vascular smooth muscle cells. Furthermore, a potassium-selective ionophore can mimic the effect of putative mitochondrial KATP channel openers. We conclude that potassium movement through KATP directly leads to ROS production by the mitochondria.