It is controversial whether or not isolated mitochondria contain an endogenous nitric oxide synthase (NOS). NOS catalyzes the production of the gaseous signaling molecule NO•(nitric oxide). During ischemia/reperfusion (IR) injury, NO•reacts with superoxide (O2•−) to form the oxidant peroxynitrite (ONOO−), which can cause tissue damage and initiate deleterious post-translational modifications of cellular proteins. The aim of this project is to detect the production of ONOO-in isolated mitochondria under conditions of oxidative/nitrosative stress, and to determine whether the ONOO-production is dependent on the presence of endogenous mitochondrial NOS. We hypothesize that isolated mitochondria contain endogenous NOS. Mitochondrial suspensions were incubated at pH 7.15, and treated with succinate, a complex II substrate, followed by menadione, a redox cycler that has been shown to generate large amounts of O2•−. In physiological buffer, L-tyrosine is oxidized by ONOO−to form the fluorescent dimer dityrosine (diTyr). We found that exposing isolated mitochondria to CaCl2, followed by succinate resulted in an increase in diTyr fluorescence signal over a 30 second period, indicating that the simulated IR injury condition increases mitochondrial ONOO− production. To verify the NOS-mediated production of ONOO−, mitochondria were incubated with the NOS inhibitors L-NAME/L-NNA, a superoxide dismutase (SOD) mimetic, TEMPOL, and PTIO, a potent global NO•scavenger. L-NAME and L-NNA both abolished the succinate and menadione-induced increase in ONOO−. The global NO• scavenger PTIO also abolished the production of ONOO-. TEMPOL partially attenuated the ONOO- levels by converting O2•− to H2O2. These results suggest that mitochondrial NOS is responsible for the production of ONOO-during simulated oxidative/nitrosative stress.