This chapter describes a luciferase-based protocol to measure adenosine triphosphate (ATP) production in isolated mitochondria ofTrypanosoma brucei. The assay represents an excellent method to characterize the functionality of isolated mitochondria. Comparing the ATP production induced by substrates for oxidative phosphorylation to the one induced by substrates for substrate-level phosphorylation allows conclusions regarding the integrity of the outer and inner mitochondrial membranes. Furthermore, the assay is a valuable tool for characterization of RNA interference cell lines suspected to affect mitochondrial functions.
