The taxonomic position of two mycolic-acid-producing actinomycetes, isolates J81 T and J82, which were recovered from activated sludge foam, was clarified. Comparative 16S rRNA gene sequence studies indicated that the organisms formed a distinct lineage within the Corynebacterineae 16S rRNA gene tree. The taxonomic integrity of this group was underpinned by a wealth of phenotypic data, notably characteristic rudimentary right-angled branching. In addition, isolate J81 T contained the following: meso-diaminopimelic acid, arabinose and galactose; N-glycolated muramic acid residues; a dihydrogenated menaquinone with eight isoprene units as the predominant isoprenologue; a fatty acid profile rich in oleic and palmitoleic acids and with relatively small proportions of myristic, stearic and tuberculostearic acids; mycolic acids with 44-52 carbons; and diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides as major polar lipids. Strain J81 T was found to have a chemotaxonomic profile that serves to distinguish it from representatives of all of the other taxa classified as belonging to the suborder Corynebacterineae. In the light of these data, it is proposed that the two isolates be classified in a novel monospecific genus. The name proposed for this taxon is Millisia brevis gen. nov., sp. nov.; strain J81 T (=DSM 44463 T =NRRL B-24424 T ) is the type strain of Millisia brevis.The taxonomy of mycolic-acid-containing actinomycetes has been clarified and extended by the application of genotypic and phenotypic procedures to representatives of established and novel taxa (Goodfellow et al., 1998a(Goodfellow et al., , 1999 Gürtler et al., 2004). Actinomycetes characterized by the presence of mycolic acids are classified in the suborder Corynebacterineae Stackebrandt et al. 1997, which encompasses the genera Corynebacterium, Dietzia, Gordonia, Mycobacterium, Nocardia, Rhodococcus, Segniliparus, Skermania, Tsukamurella and Williamsia (Butler et al., 2005;Goodfellow & Maldonado, 2006). Members of these taxa can be distinguished from one another using a combination of chemotaxonomic and morphological properties, and form distinct lineages in the Corynebacterineae 16S rRNA gene tree. Activated sludge wastewater-treatment plants with foaming problems (Soddell, 1999) are a rich source of mycolic-acid-containing actinomycetes (Lemmer & Kroppenstedt, 1984;Soddell & Seviour, 1990, 1995Goodfellow et al., 1998b;Stainsby et al., 2002) Two actinomycetes that showed rudimentary right-angled branching and which produced salmon-pink filamentous colonies were isolated from activated sludge foam by using a micromanipulator (Soddell & Seviour, 1994). Subsequent studies based on numerical taxonomic and preliminary 16S rRNA gene sequence data indicated that the two strains, isolates J81 T and J82, might represent a novel genus in the suborder Corynebacterineae , a proposition underpinned by the results of the present investigation.DNA from strains J81 T and J82 was extract...