Binding of ouabain to Na+/K+-ATPase activated multiple signal transduction pathways including stimulation of Src, Ras, p42/44 MAPKs and production of reactive oxygen species (ROS) in rat cardiac myocytes. Inhibition of either Src or Ras ablated ouabain-induced increase in both [Ca2+](i) and contractility. While PD98059 abolished the effects of ouabain on [Ca2+](i), it only caused a partial inhibition of ouabain-induced increases in contractility. On the other hand, pre- incubation of myocytes with N-acetyl cysteine (NAC) reduced the effects of ouabain on contractility, but not [Ca2+](i). Furthermore, 5- hydroxydecanoate (5-HD) blocked ouabain-induced ROS production and partially inhibited ouabain-induced increases in contractility in cardiac myocytes. Pre-incubation of myocytes with both 5-HD and PD98059 completely blocked ouabain's effect on contractility. Finally, we found that opening of mitochondrial K-ATP channel by diazoxide increased intracellular ROS and significantly raised contractility in cardiac myocytes. These new findings indicate that ouabain regulates cardiac contractility via both [Ca2+](i) and ROS. While activation of MAPKs leads to increases in [Ca2+](i), opening of mitochondrial K-ATP channel relays the ouabain signal to increased ROS production in cardiac myocytes.