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Five actinomycete strains, SK-3255T, SK-3406T, SK-3412, SK-3421 and OM-5023, were isolated using a novobiocin-containing agar medium from soil samples. These strains produced long spore chains on aerial mycelium and contained LL- and meso-diaminopimelic acids (DAPs) in the cell wall. On the basis of morphological and chemotaxonomic characteristics and phylogenetic analysis, these five strains were classified into the genus Kitasatospora. DNA-DNA hybridization and comparison of physiological characteristics revealed that strains SK-3255T and SK-3406T differed from known species. Strains SK-3406T, SK-3412 and SK-3421 were regarded as the same species. Strain OM-5023 was identified as Kitasatospora griseola. Therefore, two novel species are proposed, Kitasatospora cineracea sp. nov. and Kitasatospora niigatensis sp. nov., with the type strains K. cineracea SK-3255T (= IFO 16452T = JCM 10915T = NRRL B-23134T) and K. niigatensis SK-3406T (= IFO 16453T = JCM 10916T = NRRL B-24135T).