Mycolic acid-containing actinomycetes associated with extensive foaming in the aeration basin of the activated sludge process were isolated and analyzed by phenotypical, chemotaxonomical and phylogenetic methods. Whole cell sugar patterns of two isolates were pattern A. The nearly complete sequences of the 16S rRNA genes (rDNAs) of the isolates were determined and compared by using several tree-making algorithms. With polyphasic methods, strain SCNU1 was identified as Gordona sputi, and strain SCNU5 assigned to the genus Tsukamurella. The presence of opportunistic pathogens of chronic lung infections within foams can cause public health problems and render waste-treatment processes inefficient.