D-Serine, an important neurotransmitter, also contributes to bacterial adaptation and virulence in humans. It was reported that Pseudomonas aeruginosa PAO1 can grow on d-serine as the sole nitrogen source, and growth was severely reduced in the dadA mutant devoid of the d-alanine dehydrogenase with broad substrate specificity. In this study, the dsdA gene (PA3357) encoding a putative d-serine dehydratase was subjected to further characterization. Growth on d-serine as the sole source of nitrogen was retained in the a dagger dsdA mutant and was abolished completely in the a dagger dadA and a dagger dadA-a dagger dsdA mutants. However, when complemented by dsdA on a plasmid, the double mutant was able to grow on d-serine as the sole source of carbon and nitrogen, supporting the proposed biochemical function of DsdA in the conversion of d-serine into pyruvate and ammonia. Among d- and l-amino acids tested, only d-serine and d-threonine could serve as the substrates of DsdA, and the Km of DsdA with d-serine was calculated to be 330 mu M. Comparative genomics revealed that this cryptic dsdA gene was highly conserved in strains of P. aeruginosa, and that most strains of Pseudomonas putida possess putative dsdCAX genes encoding a transcriptional regulator DsdC and a d-serine transporter DsdX as in enteric bacteria. In conclusion, this study supports the presence of a cryptic dsdA gene encoding a functional d-serine dehydratase in P. aeruginosa, and the absence of dsdA expression in response to exogenous d-serine might be due to the loss of regulatory elements for gene activation during evolution.