Site-directed mutagenesis of specific residues in bacteriophage T4 lysozyme is shown to result in changes in the emission spectra of the tryptophan residues of this protein. In some cases a significant red-shift is observed. This is interpreted in terms of enhanced dielectric relaxation due to fluctuations that expose a buried residue to the aqueous solvent. For substitutions at position 146, the spectral shift is strongly correlated with the rate of a specific proteolytic digestion of the T4 lysozyme by trypsin as determined by Signor, Dalzoppo, and Schellman. In cases where a spectral shift is observed there is also an enhancement of internal mobility of a tryptophan residue as indicated by the amplitude of a short correlation time component of the anisotropy decay. All of these spectral and enzymatic susceptibility effects are reversed by introduction of a disulfide linkage spanning the two lobes of the protein. The interpretation of these results in terms of molecular dynamics is discussed. The effect of mutational changes on the average fluorescence lifetime and quantum yield of tryptophan fluorescence is also discussed in terms of collisional quenching of tryptophan residues by neighboring groups.