Partial sequences of the 16S rRNA genes of the type strains of nine species of the genus Nocardia were determined following the isolation and cloning of the amplified genes. These sequences were aligned with the sequences of representatives of the genera Corynebacterium, Gordona, Mycobacterium, Rhodococcus, and Tsukamurella, and phylogenetic trees were inferred by using the Fitch-Margoliash and neighbor-joining methods. The genus Nocardia formed a distinct clade that was most closely associated with the genus Rhodococcus. The average level of sequence similarity found among the type strains of the Nocardia species was 97.2 +/- 0.7%. Two sublines were recognized within the Nocardia clade; one encompassed Nocardia asteroides and related species, and the other encompassed Nocardia otitidiscaviarum and allied taxa. Separation of the two sublines is based on differences in helix 37-1. The results of isoprenoid quinone analyses provided evidence that nocardiae can be distinguished from all other actinomycete taxa on the basis of their characteristic menaquinone profiles. Nocardiae typically contain hexahydrogenated menaquinones with eight isoprene units in which the two end units are cyclized.