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-L30 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.