Tryptophyl residues in proteins absorb at longer wavelengths than tyrosyl residues, thus the tryptophyl fluorescence can be selectively excited. In addition, tryptophyl residues have a fluorescence maximum at much longer wavelengths than tyrosyl residues and are the predominant source of fluorescence in the long-wavelength region. The contribution of tyrosyl fluorescence to protein fluorescence can be determined by exploiting these spectral properties. The fluorescence of the tyrosyl residues in native human serum albumin is different from the fluorescence of N-acetyl-L-tyrosinamide; the spectral maximum is at a longer wavelength, and the spectral width is greater. This difference is caused by a second component with a maximum at 345 nm. The excitation spectrum of the 345-nm component is similar to that of the normal 304-nm tyrosyl component. The 345-nm component is largely absent from denatured serum albumin. An excited singlet state protolysis from the buried tyrosyl residues explains the appearance of the 345-nm component. A possible acceptor base is an amino group of buried lysyl residue.