The pathways involved in the reduction of various tetrazolium salts catalyzed by beef-heart mitochondria were investigated. It was found that the reduction of 2,2′,5′-tetraphenyl-3,3′-(4,4′-biphenylene)ditetrazolium chloride, 2- p-iodophenyl-3- p-nitrophenyl-5-phenyltetrazolium chloride, and 2,5-diphenyl-3-α-naphthyletrazolium chloride with succinate as substrate were dependent on CoQ and were largely sensitive to antimycin. These tetrazoles were not reduced by the reduced forms of the isolated cytochromes. Hence these interact somewhere at or beyond the antimycin site and before cytochromes c 1 and c. 2,2′-di- p-nitrophenyl-5,5′-diphenyl-3,3′-(3,3′-dimethoxy-4,4′- biphenylene)ditetrazolium chloride reduction by succinate is not CoQ-dependent and appears to occur at a site responsible for CoQ reduction. Reduction of tetrazoles by reduced diphosphopyridine nucleotide would appear to take place at the flavoprotein level. Evidence indicating that different requirements exist for the reduction of K 3 and CoQ is also presented. Data on the lipid requirements for tetrazole reduction in lipid-depleted mitochondrial preparations are given. A simple assay procedure is described for the enzymic reduction of tetrazolium salts.