The inability of cells and microorganisms to reduce the colourless electron acceptor triphenyltetrazolium chloride (TTC) to a red formazan precipitate is commonly used as a means of screening for cells that have a dysfunctional respiratory chain. The site of reduction of TTC is often stated to be at the level of cytochrome c oxidase where it is assumed to compete with oxygen for reducing equivalents. However, we show here that TTC is reduced not by cytochrome c oxidase but instead by dehydrogenases, particularly complex I, probably by accepting electrons directly from low potential cofactors. The reduction rate is fastest in coupled membranes because of accumulation in the matrix of the positively charged TTC+ cation. However, the initial product of TTC reduction is rapidly reoxidised by molecular oxygen, so that generation of the stable red formazan product from this intermediate occurs only under strictly anaerobic conditions. Colonies of mutants defective in cytochrome oxidase do not generate sufficiently anaerobic conditions to allow the intermediate to form the stable red formazan. This revision of the mode of interaction of TTC with respiratory chains has implications for the types of respiratory-defective mutants that might be detected by TTC screening.