The methods described serve to illustrate not only the importance of measuring NȮ. thresholds in mitochondria and cells, but also highlight many of the considerations that must be taken when making such measurements. These include the presence of O2, the source of NȮ., the precise design of the apparatus, and experimental conditions, such as buffer composition, light level, and temperature. When all these factors are considered, accurate interpretations of the role of NȮ. in controlling mitochondrial function can be made. Such experiments can provide insight into the intricate nature of the interaction between NȮ. and mitochondria and the variations in this system that can occur under both physiological and pathological conditions.