The binding of riboflavin, 3-methylriboflavin, and lumiflavin to the apoprotein of egg white flavoprotein was studied by fluorometry, spectrophotometry, and the circular dichroism (CD) technique. The fluorescence of the flavins was completely quenched upon their binding to the apoprotein, whereas 86% of the fluorescence of the protein was quenched on its binding to riboflavin or 3-methylriboflavin, and 77% on its binding to lumiflavin. The association constants of the flavin-apoprotein binding, obtained from fluorometric titrations, were 5.0x108M−1 for riboflavin, 4.0 x 108M−1 for 3-methylriboflavin, and 2.4 x107M−1 for lumiflavin. The visible absorption spectra of all the flavins were modified on their binding to the apoprotein. The CD spectra of the riboflavin- and 3-methylriboflavin-apoprotein complexes were similar in the range of wavelengths investigated (200–550 nm), but differed from the CD spectrum of the lumiflavin-apoprotein complex. The difference absorption spectra in the near-ultraviolet region resulting from the binding of the flavins to the apoprotein indicated perturbations of the spectra of both the flavin and aromatic amino acid residues of the protein. These spectral perturbations suggested that the aromatic groups became less accessible to solvent or interacted with the flavin. The comparison of the CD spectra of the flavinapoprotein complexes with the spectrum of the apoprotein also supported this view. The CD data in the region from 200 to 225 nm indicated that the conformation of the polypeptide backbone of the apoprotein was hardly affected by the binding of the flavins.