Abstract 1. 1. Excitation difference spectra techniques have been utilized to study energy transfer in enzymes. Definite evidence for tyrosine to tryptophan energy transfer is found in alcohol:NAD oxidoreductase (EC 1.1.1.1) and α-1,4-glucan-4-glucano-hydrolase ( Bacillus subtilus ) (EC 3.2.1.1), but not in α-chymotrypsin (EC 3.4.4.5), papain (EC 3.4.4.10), α-1,4-glucan-4-glucanohydrolase ( Aspergillus oryzae ) (EC 3.2.1.1), pepsin (EC 3.4.4.1), and carboxypeptidase A (EC 3.4.2.1). 2. 2. The phosphorescence excitation spectra provide direct experimental evidence that the enzyme emission in the region from 350 to 390 nm in due to tyrosine residues. Futhermore, these data suggest that the emitting tyrosine residues in the enzymes appear to be in an aqueous-like environment and therefore presumbly exposed to the solvent. 3. 3. Phosphorescence excitation difference spectra indicate that those tyrosine residues which are responsible for transferring energy to tryptophan residues are probably in a non-aqueous environment. 4. 4. Neither the relative tyrosine/tryptophan phosphrescence yields, nor the absolute yields in the enzymes could be simply correlated with the properties of the free amino acids.