The phylogenetic relationships among 30 mycolic acid-containing wall chemotype IV actinomycete strains and 12 strains belonging to allied taxa were examined by determining the amino acid sequences of the ribosomal AT-L30 proteins of these organisms. Sequencing 20 N-terminal amino acids of AT-L30 preparations revealed that the members of the genera containing mycolic acid-containing actinomycetes form two clusters; the first cluster contains the genera Nocardia, Rhodococcus, Gordona, and Tsukamurella, and the second cluster contains the genera Corynebacterium and Mycobacterium. The genus Nocardia was placed in a clade containing the genus Rhodococcus. The data showed that Tsukamurella paurometabolum is closely related phylogenetically to the genus Gordona. The phylogenetic clusters identified were entirely consistent with the proposal of Goodfellow that the family Nocardiaceae should encompass the mycolate-containing, cell wall type IV actinomycete genera Nocardia, Rhodococcus, Gordona, and Tsukamurella. The genera Actinomyces and Micrococcus exhibited AT-UO amino acid sequence characteristics intermediate between those of actinomycetes and those of typical eubacteria. The genera Nocardia, Gordona, Mycobacterium, Actinoplanes, and Micromonospora were each a taxon that consisted of phylogenetically coherent species. In contrast, the genera Rhodococcus and Corynebacterium are taxa that consist of phylogenetically distantly related species. In general, my results are consistent with previous 16s rRNA sequencing results, but significant differences were also found. My data, together with previous AT-L30 sequencing data, show that phylogenetic relationships among taxa can be determined by using markers other than the ribosomal gene sequences.Microorganisms containing mycolic acids in their cell walls have many properties in common (13) and form a recognizable suprageneric group (28,49,52). These organisms have chemotype IV walls (26) and can be assigned to the genera Coiynebacterium, Gordona, Mycobacterium, Nocardia , Rhodococcus, and Tsukamurella by using a combination of chemical and morphological markers (12). Nocardiae produce abundant or sparse aerial mycelia, but corynebacteria do not produce mycelia. Mycobacteria and rhodococci do not usually form aerial hyphae, with a few exceptions. The taxonomy of the actinomycetes containing mycolic acids has undergone significant revisions based on data obtained by chemical, numerical, and molecular systematic methods; such revisions have occurred in the genera Rhodococcus (11-13, 15), Corynebacterium (27), Gordona (51), Tsukamurella (4), and Mycobacterium (17,18,46,60). The genus Nocardia is the type genus of the family Nocardiaceae. The genus Gordona is characterized by the absence of mycelia and slight or strong acid fastness, and members of this genus have characteristics intermediate between those of the genus Nocardia and those of the genus Mycobactenurn (54). The taxonomy of the genus Gordona and the related genus Tsukamurella has been clarified by using chemical, m...