Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride) is widely used as a redox cycler to stimulate superoxide production in organisms, cells, and mitochondria. Paraquat is also used to induce symptoms of Parkinson's disease in experimental models of this neurodegenerative disorder. Paraquat causes extensive mitochondrial oxidative damage, and in mammalian systems, complex I of the respiratory chain has been identified as the major site of superoxide production by paraquat. Although much progress has been made at explaining how paraquat interacts with mitochondria, several aspects remain to be clarified-most notably the pathway of paraquat uptake into mitochondria. This chapter describes methods for further investigating the interaction of paraquat with mitochondria and also provides practical information for the general use of paraquat as a superoxide generator and agent of oxidative stress. The techniques covered include the detection and quantitation of the paraquat dication and the paraquat monocation radical (by electron paramagnetic resonance, spectrophotometry, and with an ion-selective electrode); assays for measuring paraquat-induced superoxide production by intact mitochondria or mitochondrial membranes (including aconitase inactivation, and coelenterazine chemiluminescence); methods for assessing paraquat uptake by mitochondria; and screens for identifying paraquat sensitivity or resistance in yeast mutants.