Superoxide is the proximal reactive oxygen species (ROS) produced by the mitochondrial respiratory
chain and plays a major role in pathological oxidative stress and redox signaling. While there are tools to
detect or decrease mitochondrial superoxide, none can rapidly and specifically increase superoxide
production within the mitochondrial matrix. This lack impedes progress, making it challenging to assess
accurately the roles of mitochondrial superoxide in cells and in vivo. To address this unmet need, we
synthesized and characterized a mitochondria-targeted redox cycler, MitoParaquat (MitoPQ) that comprises
a triphenylphosphonium lipophilic cation conjugated to the redox cycler paraquat. MitoPQ accumulates
selectively in the mitochondrial matrix driven by the membrane potential. Within the matrix,
MitoPQ produces superoxide by redox cycling at the flavin site of complex I, selectively increasing superoxide
production within mitochondria. MitoPQ increased mitochondrial superoxide in isolated mitochondria
and cells in culture a thousand-fold more effectively than untargeted paraquat. MitoPQ was
also more toxic than paraquat in the isolated perfused heart and in Drosophila in vivo. MitoPQ enables the
selective generation of superoxide within mitochondria and is a useful tool to investigate the many roles
of mitochondrial superoxide in pathology and redox signaling in cells and in vivo.