Photoreduced flavins react very rapidly with molecular oxygen to give the superoxide anion. This radical is highly reactive with cytochrome c and p-nitro blue tetrazolium salts. The reduction of these electron acceptors under aerobic conditions is strongly inhibited by catechols such as 1,2-dihydroxybenzene-3,5-disulfonate (Tiron) and 3,4-dihydroxybenzoic acid. Tiron also is essential for the photosensitized reoxidation of ferrocytochrome c in the presence of flavins and oxygen. These findings support a proposal that catechols react rapidly with superoxide anion to yield a new oxidizing species. This fast reaction effectively prevents the oxygen radical from participating in slower oxidation–reduction processes. Inhibition by Tiron of the oxygen-dependent catalytic reduction of electron acceptors is therefore indicative of the participation of enzymatically generated superoxide anion. The proposed explanation of the observed phenomena does not require interaction of the catechol inhibitor with the iron–sulfur chromophores of superoxide-forming metalloflavoproteins.