The phylogenetic relationships of the genera belonging to the family Pseudonocardiaceae were examined by a novel approach, amino acid sequencing of ribosomal AT-L30 proteins. The results of partial amino acid sequencing of AT-L30 preparations revealed that the members of the family Pseudonocardiaceae are divided into four clusters; the first cluster contains the genus Actinopolyspora, the second cluster contains the genus Saccharopolyspora, the third cluster contains the genus Amycolatopsis, and the fourth cluster contains the genera Amycolata, Pseudonocardia, Saccharomonospora, and Kibdelosporangium, indicating a close phylogenetic relationship between the genera Amycolata and Pseudonocardia. The genus Actinokineospora is closely related to the genus Saccharothrix, and these two genera formed a cluster separate from the clusters for the genera of the Pseudonocardiaceae. These results agree in almost all respects with previous 16S rRNA sequencing work by Embley et al. (T. M. Embely, J. Smida, and E. Stackebrandt, Syst. Appl. Microbiol. 11:44-52, 1988) and Warwick et al. (S. Warwick, T. Bowen, H. McVeigh, and T. M. Embley, Int. J. Syst. Bacteriol. 44:293-299, 1994), thus supporting the proposal of Warwick et al. that the genera Amycolata and Pseudonocardia should be combined in an emended genus, Pseudonocardia. However, a discrepancy was found between the present study and that of Warwick et al. In the present study, the Nocardia-Rhodococcus group and the Saccharothrix-Actinokineospora group were both recovered within the clade for the family Pseudonocardiaceae.