The homologous gene of D-amino acid oxidase (DAO) in prokaryotic organisms is predominantly found in a group of bacteria called the Actinobacteria. We have analyzed the DAO of the model actinomycete Streptomyces coelicolor and the effect of D-amino acids on this bacterium. When expressed in Escherichia coli, the translated product of the putative dao gene of this bacterium exhibited oxidase activity against neutral and basic D-amino acids, with a higher activity toward D-valine and D-isoleucine, but not to their corresponding L-amino acids. This substrate specificity was largely different from that of the DAO of the actinobacterium Arthrobacter protophormiae. The gene message and DAO activity were constitutively detected in S. coelicolor cells, and unlike eukaryotic DAOs, the presence of a D-amino acid did not significantly induce expression. The D-amino acids that were a good substrate for S. coelicolor DAO inhibited cell growth, delayed morphological development and affected cell morphology, but they did not inhibit biofilm formation. Disruption of the dao gene had no effect on the morphology and morphological development of S. coelicolor cells, the assimilation of D-valine or the sensitivity to growth inhibition by D-valine under the experimental conditions, showing that in this bacterium DAO does not play a significant role in either morphological development or the assimilation and detoxification of D-amino acids.