Objective: Acetylcholine (ACh) mimics ischemic preconditioning (PC) and therefore protects the heart against lethal ischemia. Steps common to both ischemic and drug-induced PC are opening of mitochondrial KATP channels (mito KATP) and generation of reactive oxygen species (ROS). The aim of this study was to test whether ACh-induced ROS production could be seen in a vascular smooth muscle cell line, and, if so, to investigate the underlying signaling pathway. Methods: Mitochondrial ROS generation was quantified by measuring changes in fluorescence of ROS-sensitive intracellular markers in vascular smooth muscle cells (A7r5). Results: Fluorescence, and, therefore, ROS production, was increased to 197.5±8.5% of baseline after 45 min of exposure of cells to 2 mM ACh (Pi with pertussis toxin (PTX) (90.5±4.4%), implicating a receptor-mediated rather than non-specific effect of ACh. The increased fluorescence induced by ACh was also abrogated by the free radical scavenger N-(2-mercaptopropionyl) glycine (104.2±10.1%), documenting that ROS were indeed the cause of the enhanced fluorescence. Both diazoxide, a KATP channel opener, and valinomycin, a potassium ionophore, also significantly increased ROS production, and these effects were not blocked by PTX, while the KATP channel closer 5-hydroxydecanoate blocked ACh-induced ROS production (92.3±3.8%). These results suggest ROS production is directly influenced by KATP activity and K+ movements in the cell. The tyrosine kinase inhibitor genistein (102.8±6.6%) and the phosphatidylinositol 3 (PI3)-kinase inhibitor wortmannin (90.7±4.1%) also inhibited the ability of ACh to increase ROS production. Conclusion: The signaling pathway by which ACh leads to ROS generation in A7r5 cells involves a muscarinic surface receptor, a pertussis toxin-sensitive G protein, PI3-kinase, at least one tyrosine kinase, and a 5-hydroxydecanoate (5-HD)-dependent KATP (presumably that in mitochondria).