Ketosteroid isomerase (KSI) from Pseudomonas putida blotype B is a homodimeric enzyme catalyzing an allylic rearrangement of Δ5-3-ketosteroids at rates comparable with the diffusion-controlled limit. The tyrosine triad (Tyr14···Tyr55···Tyr30) forming a hydrogen-bond network in the apolar active site of Kcat has been characterized in an effort to identify the roles of the phenyl rings in catalysis, stability, and unfolding of the enzyme. The replacement of Tyrl4, a catalytic residue, with serine resulted in a 33-fold decrease of kcat, while the replacements of Tyr30 and Tyr55 with serine decreased kcat by 4- and 51-fold, respectively. The large decrease of kcat for Y55S could be due to the structural perturbation of α-helix A3, which results in the reorientation of the active-site residues as judged by the crystal structure of Y55S determined at 2.2 Å resolution. Consistent with the analysis of the Y55S crystal structure, the far-UV circular dichroism spectra of Y14S, Y30S, and Y55S indicated that the elimination of the phenyl ring of the tyrosine reduced significantly the content of α-helices. Urea-induced equilibrium unfolding experiments revealed that the ΔGUH2O values of Y14S, Y30S, and Y55S were significantly decreased by 11.9, 13.7, and 9.5 kcal/mol, respectively, as compared with that of the wild type. A characterization of the unfolding kinetics based on ΦU-value analysis indicates that the interactions mediated by the tyrosine triad in the native state are very resistant to unfolding. Taken together, our results demonstrate that the internal packing by the phenyl rings in the active-site tyrosine triad contributes to the conformational stability and catalytic activity of KSI by maintaining the structural integrity of the α-helices.