Reactive oxygen species (ROS) play a vital role in cell signaling and redox regulation, but when present in excess, lead to numerous pathologies. Detailed quantitative characterization of mitochondrial superoxide anion (O;-2 production in fetal pulmonary artery endothelia cells (PAECs) has never been reported. The aim of this study is to assess mitochondrial O;-2 production in cultured PAECs over time using a novel quantitative optical approach. The rate, the sources, and the dynamics of O;-2 production were assessed using targeted metabolic modulators of the mitochondrial electron transport chain (ETC) complexes, specifically an uncoupler and inhibitors of the various ETC complexes, and inhibitors of extra-mitochondrial sources of O;-2. After stabilization, the cells were loaded with nanomolar mitochondrial-targeted hydroethidine (Mito-HE, MitoSOX) online during the experiment without washout of the residual dye. Timelapse fluorescence microscopy was used to monitor the dynamic changes in O;-2 fluorescence intensity over time in PAECs. The transient behaviors of the fluorescence time course showed exponential increases in the rate of O;-2 production in the presence of the ETC uncoupler or inhibitors. The most dramatic and the fastest increase in O;-2 production was observed when the cells were treated with the uncoupling agent, PCP. We also showed that only the complex IV inhibitor, KCN, attenuated the marked surge in O;-2 production induced by PCP. The results showed that mitochondrial respiratory complexes I, III and IV are sources of O;-2 production in PAECs, and a new observation that ROS production during uncoupling of mitochondrial respiration is mediated in part via complex IV. This novel method can be applied in other studies that examine ROS production under stress condition and during ROS-mediated injuries in vitro.