Tyrosine 265 (Y265) of Bacillus stearothermophilus is believed to serve as a catalytic base specific to the L-enantiomer of a substrate amino acid by removing (or returning) an α-hydrogen from (or to) the isomer on the basis of the X-ray structure of the enzyme [Stamper, C. G., Morollo, A. A., and Ringe, D. (1998) Biochemistry 37, 10438-10443]. We found that the Y265→Ala mutant (Y265A) enzyme is virtually inactive as a catalyst for alanine racemization. We examined the role of Y265 further with β-chloroalanine as a substrate with the expectation that the Y265A mutant only catalyzes the α, β-elimination of the D-enantiomer of β-chloroalanine. However, L-β-chloroalanine also served as a substrate; this enantiomer was rather better as a substrate than its antipode. Moreover, the mutant enzyme was as equally active as the wild-type enzyme in the elimination reaction. These findings indicate that Y265 is essential for alanine racemization but not for β-chloroalanine elimination.