Paraquat (PQ) is a prototypical redox cycling agent commonly used experimentally to generate reactive oxygen species and oxidative stress. Recently, PQ has also come under investigation as a potential environmental neurotoxin associated with increased risk for neurodegenerative disease developing after chronic exposure. The interactions of PQ with mitochondria remain an important aspect of its toxicity, particularly in the brain, although the underlying mechanisms are relatively uncharacterized. Here, we describe the basic measurement of PQ-induced hydrogen peroxide (H(2)O(2)) production in isolated brain mitochondria by use of two independent methods, polarography and fluorometry. The advantages of the use of these two independent methods include the capability to validate results and overcoming limitations in the use of either method exclusively. These simplified in vitro techniques for measurement of mitochondrial-generated H(2)O(2) can be easily applied to other tissues and models.