We have measured the steady-state phosphorescence and decay times of wild-type λcI repressor and compared it with that of a modified λcI repressor in which > 95% of the tryptophans were replaced with 5-hydroxy-L-tryptophan (5-OHTrp). The wild-type and 5-OHTrp-λcI repressors are spectroscopically distinct such that we can selectively excite the 5-OHTrp-λcI even in the presence of a 15-fold molar excess ofN-acetyltryptophanamide (NATrpA). The phosphorescence band of wild-type λcI is red-shifted by 3 nm relative to NATrpA, characteristic of buried tryptophan. Similarly, the phosphorescence of 5-OHTrp-λcI repressor is red-shifted relative to the model, 5-OHTrp, showing that according to the phosphorescence, the modified repressor is structurally indistinguishable from the native repressor. While the phosphorescence decay of both NATrpA and 5-OHTrp are single exponentials, the decay of both wild-type and 5-OHTrp-λcI repressors is complex, requiring three decay components whose fractional contributions to the phosphorescence are the same for both repressors. Because the 5-OHTrp phosphorescence can be excited at wavelengths outside the absorbance range of tryptophan and DNA, a protein spectrally enhanced with this emitter will aid the investigations of protein-protein or protein-DNA interactions.