The 16S-23S internally transcribed spacer (ITS) sequences were analysed to clarify inter- and intraspecific relationships among strains of the genus Nocardioides and the relationship between two Aeromicrobium species. The 16S-23S ITS regions from 33 Nocardioides strains, two Aeromicrobium species and Terrabacter tumescens were sequenced directly after polymerase chain reaction (PCR0 amplification and gamma exonuclease treatment. The genomes of some Nocardioides strains included two types of 16S-23S ITS sequences. The sizes of the 16S-23S ITS sequences of Nocardioides strains ranged from 328 to 539 bp. The 16S-23S ITS sequences of Aeromicrobium erythreum NSP37T, Aeromicrobium fastidiosum NSP38T and T. tumescens NSP39T were 349, 355 and 386 bp long, respectively. Nucleotide similarity among 16S-23S ITS sequences of Nocardioides albus strains and of Nocardioides simplex strains was 84.1-100% and 97.7-100%, respectively. The 16S-23S ITS sequence of Nocardioides luteus was identical to that of 'Nocardioides fulvus' NSP32T and was only 1 bp different from that of 'Nocardioides flavus' strains. However, the 16S-23S ITS sequences of 'N. fulvus' NSP33 showed only a low degree of similarity to 'N. fulvus' NSP32T (54.8%). The degree of 16S-23S ITS similarity between N. luteus NSP20T and N. ablus strains ranged from 85 to 93%. The mean nucleotide similarity values between the type strains of validly described Nocardioides species were highly divergent at 68:1 +/- 16.8%. The two Aeromicrobium species showed a level of 16S-23S ITS similarity of 71.2%. In this study, 16S-23S ITS sequences of the members of the genera Nocardioides and Aeromicrobium were useful for inferring the relationships between closely related strains and species. However, they were not found to be appropriate for elucidating the phylogenetic relationships between distantly related organisms at the genus level.