Three novel bacterial strains were isolated from a soil sample collected in Japan by culture on a GPM agar plate supplemented with superoxide dismutase and catalase. The strains were Gram-positive, catalase-positive, non-motile bacteria with L-ornithine as a diagnostic diamino acid of the peptidoglycan. The acyl type of the peptidoglycan was N-glycolyl. The major menaquinones were MK-12, 13 and 14. Mycolic acids were not detected. G+C contents of the DNA were in the range 69-71 mol%. Comparative 16S rRNA gene sequence analysis revealed that the isolates belonged to the genus Microbacterium and were closely related to Microbacterium terregens, Microbacterium aurum, Microbacterium koreense, Microbacterium schleiferi and Microbacterium lacticum. However, M. aurum, M. koreense and M. lacticum clearly differed from the isolated strains based on the presence of L-lysine as the cell-wall diamino acid and various other chemotaxonomic characteristics. Levels of DNA-DNA relatedness showed that the isolated strains represented three separate genomic species. Based on both phenotypic and genotypic data, the following novel species of the genus Microbacterium are proposed: Microbacterium deminutum sp. nov. (type strain KV-483(T)=NRRL B-24453(T)=NBRC 101278(T)), Microbacterium pumilum sp. nov. (type strain KV-488(T)=NRRL B-24452(T)=NBRC 101279(T)) and Microbacterium aoyamense sp. nov. (type strain KV-492(T)=NRRL B-24451(T)=NBRC 101280(T)).