Three tyrosine residues but no tryptophan exist in the ribose-binding protein (RBP) of Escherichia coli. In order to assess the contribution of each tyrosine to the fluorescence properties, mutants were constructed by site-directed mutagenesis to replace tyrosines at 32, 115, and 261 by phenylalanines. The mutant proteins were functional as confirmed by in vivo tactic response and by their ability to bind to ribose. The fluorescence emission spectra of the native proteins purified from the various tyrosine mutants were measured from emission scans with a peak at 303 nm. The tyrosines, at positions 32, 115, and 261, contribute 10.0, 69.6, and 23.4%, respectively, to the total intensity of fluorescence. In completely unfolded polypeptide, these tyrosines have almost the same intensities of fluorescence, indicating that the fluorescence from tyrosines at 32 and 261 are considerably quenched in the folded, native protein.