A novel actinobacterium, strain N9999(T), was isolated from soil and its taxonomic position determined using a polyphasic approach. The organism formed abundant aerial hyphae that differentiated into spherical spore vesicles. The cell wall contained meso-diaminopimelic acid; the whole-cell sugars were galactose, glucose, mannose, madurose and ribose; the predominant menaquinones MK-9 (H(2)) and MK-9 (H(4)); the major phospholipids phosphatidylethanolamine, diphosphatidylglycerol, a phosphaglycolipid and phosphatidylinositol mannosides; while the cellular fatty acids were rich in iso-C(14:0), C(15:0), cis-9-C(17:1), iso-C(16:0) and 10-methyl C(17:0) components. Phylogenetic analyses based on an almost complete 16S rRNA gene sequence indicated that strain N9999(T) was closely related to a group that consisted of Streptosporangium pseudovulgare DSM 43181(T) and Streptosporangium nondiastaticum DSM 43848(T). However, DNA-DNA relatedness and phenotypic data demonstrated that strain N9999(T) was clearly distinguished from all closely related Streptosporangium species. The combined genotypic and phenotypic data demonstrate conclusively that the isolate should be classified as a new species of Streptosporangium.