Y238, one of the very few conserved residues in the active site of d-amino acid oxidases (DAAO), was mutated to phenylalanine and serine in the enzyme from the yeast Rhodotorula gracilis. The mutated proteins are catalytically competent thus eliminating Tyr238 as an active-site acid/base catalyst. Y238F and Y238S mutants exhibit a threefold slower turnover on d-alanine as substrate, which can be attributed to a slower rate of product release relative to the wild-type enzyme (a change of the rate constants for substrate binding was also evident). The Y238 DAAO mutants have spectral properties similar to those of the wild-type enzyme but the degree of stabilization of the flavin semiquinone and the redox properties in the free form of Y238S are different. The binding of the carboxylic acid competitive inhibitors and the substrate d-alanine are changed only slightly, suggesting that the overall substrate binding pocket remains intact. In agreement with data from the pH dependence of ligand binding and with the protein crystal structure, site-directed mutagenesis results emphasize the importance of residue Y238 in controlling access to the active site instead of a role in the substrate/ligand interaction.