Abstract The function of the highly conservative sequence —G(344)-F-S-G-H-G-F-K-F(352)— in the carboxyl terminus of sarcosine oxidase was investigated using site-directed mutagenesis. When H-348 was substituted with uncharged amino acids, the K m values of sarcosine oxidase markedly increased, although the k cat values remained the same as that of the wild-type enzyme. The kinetic parameters obtained with other mutations also suggested that the conservative sequence acts as the substrate-binding site. When K-351 was replaced by Ala, the mutant K351A could not bind the coenzyme FAD (flavin adenine dinucleotide). This result suggested that K-351 interacts with the FAD-binding site of the amino-terminal region. The enzymic activities of the mutants H348Q and H348A were lost at neutral and acidic pH as a result of the disappearance of the positive charge on H-348.