Experiments have been performed to probe the flavin adenine dinucleotide (FAD) binding region in Escherichia coli pyruvate oxidase. This enzyme functions as a membrane-associated flavoprotein coupled to the aerobic E. coli respiratory chain. The FAD moiety is noncovalently bound to pyruvate oxidase and can be removed reversibly to form apopyruvate oxidase. The addition of free FAD to apoenzyme results in the stoichiometric re-formation of the active flavoprotein. Using this technique, synthetic analogs of FAD were substituted in the flavin binding site and used as structural probes. Spectral analysis indicates that the benzoquinoid forms of 8-mercapto-FAD and 6-hydroxy-FAD are stabilized in the enzyme-binding site. This is consistent with the fact that the native flavoprotein forms a red (anion) radical upon photoreduction. These data suggest that the isoalloxazine ring may be poised for reduction via position N-5 by a carbanionic intermediate. The alpha-carbanion of hydroxyethylthiamin pyrophosphate, formed following the decarboxylation of pyruvate, is a likely candidate. The highly resolved visible spectrum of the native flavoprotein suggests that the flavin is buried in a hydrophobic environment. Reactivity studies using 8-chloro-FAD-pyruvate oxidase and 2-thio-FAD-pyruvate oxidase suggest that the C-8 position and C-2 position of the isoalloxazine ring may not be accessible to the solvent. Spectral perturbations observed with 6-hydroxy-FAD-pyruvate oxidase indicate, however, that the isoalloxazine C-6 position may be located near the binding site for the cofactor thiamin pyrophosphate. Restrictions to the accessibility of the active site of the enzyme are suggested by the fact that sulfite does not form an adduct with the flavin in the native enzyme.