Δ5-3-Ketosteroid isomerase (EC 126.96.36.199) of Pseudomonas testosteroni converts Δ5-3-ketosteroids to Δ4-3-ketosteroids via an enolic intermediate. Site-specific mutagenesis has identified Tyr-14 and Asp-38 as the catalytically essential general acid and base, respectively. Three tyrosine residues (Tyr-14, Tyr-55, and Tyr-88) are the only significant fluorophores in the wild-type isomerase. Recent studies of the steady-state fluorescence of the wild-type enzyme and all six mutant enzymes in which one or two tyrosine residues have been mutated to phenylalanine show that the fluorescence intensity of Tyr-14 is very high, that of Tyr-88 is very low, and that of Tyr-55 is intermediate and comparable to that of N-acetyltyrosine amide in solution (Li, Y.-K., Kuliopulos, A., Mildvan, A. S., & Talalay, P. (1993) Biochemistry 32, 1816–1824). Extension of these experiments by time-resolved fluorescence and fluorescence anisotropy measurements demonstrates that Tyr-14, which is in a hydrophobic environment, has an unusually long fluorescence lifetime (4.6 ns) as compared to Tyr-55 (2.0 ns) or Tyr-88 (0.8 ns) and to most protein tyrosine residues (0.2–2 ns). The Forster distances obtained from the absorption and emission of these tyrosines predict that total quenching of Tyr-14 fluorescence by Tyr-55, and to a lesser degree by Tyr-88, would occur if their orientations were favorable. The lack of efficient quenching of Tyr-14 fluorescence by Tyr-55 implies that Tyr-14 and Tyr-55 are oriented unfavorably for efficient resonance energy transfer and that this orientation is rigid on the time scale of picoseconds to nanoseconds. The rigidity of Tyr-14 and Tyr-55 with respect to one another is also confirmed by time-resolved fluorescence anisotropy at 20 and 40 °C, where only one correlation time corresponding to the global motion of the protein is resolved. Circular dichroism (CD) measurements on isomerase denatured by heat or guanidine hydrochloride have also confirmed that changes of Tyr-14 fluorescence in an isolated environment are tightly coupled to the changes in the protein structure and dynamics.