Objectives: Ischemic preconditioning (PC) reduces myocardial infarction by a mechanism that involves opening of mitochondrial ATP-dependent potassium channels (mK A T P ), reactive oxygen species (ROS), and possibly activation of p38 mitogen-activated protein kinase (p38 MAPK). The actual order of these steps, however, is a matter of current debate. This study examined whether protection afforded by menadione, which protects by causing mitochondria to produce ROS, requires mK A T P opening. In addition, we tested whether protection from anisomycin, a p38 MAPK activator, is dependent on ROS production. Methods and Results: Isolated, buffer-perfused rat hearts were pretreated with menadione, and infarction was assessed after 30 min of regional ischemia and 120 min of reperfusion. Menadione reduced infarction in a dose-dependent manner with an EC 5 0 of 270 nM. Menadione's infarct-limiting effect was insensitive to 200 μM 5-hydroxydecanoate (5HD), an mK A T P channel blocker, whereas protection by diazoxide and PC were blocked by 5HD. Anisomycin caused hearts to resist infarction and this protective effect was abrogated by SB203580, a p38 MAPK inhibitor, and 2-mercaptopropionylglycine (MPG), a free radical scavenger. Conclusions: These results indicate that mK A T P opening occurs upstream of mitochondrial ROS generation in the protective pathway. Furthermore, protection afforded by anisomycin was p38 MAPK- and ROS-dependent.