Pyruvate oxidase of Escherichia coli possesses a reactive cysteine residue believed to be associated with the thiamin pyrophosphate (TPP) binding site. This residue is not reactive in the presence of TPP. Exposure of the enzyme to cysteine-directed fluorescent reagents results in the formation of fluorescent protein conjugates. Although these reagents do not react solely with the TPP-protectable cysteine residue, the fluorescence emission spectrum of a probe attached to this residue can be obtained by a difference technique. It was determined that the fluorescence emission of probes at the TPP-protectable site is very low due to energy transfer to the FAD coenzyme and that this fluorescence is greatly enhanced upon reduction or extraction of the flavin. Application of fluorescence energy transfer theory enabled the determination of an upper limit for the distance between the probes at the TPP-protectable site and the flavin adenine dinucleotide (FAD) (roughly 20 A). Thus, the TPP binding site and the FAD coenzyme are likely in close proximity.