The phylogenetic position of the Mollicutes has been re-examined by using phosphoglycerate kinase (Pgk) amino acid sequences. Hitherto unpublished sequences from Mycoplasma mycoides subsp. mycoides, Mycoplasma hyopneumoniae and Spiroplasma citri were included in the analysis. Phylogenetic trees based on Pgk data indicated a monophyletic origin for the Mollicutes within the Firmicutes, whereas Bacilli (Firmicutes) and Clostridia (Firmicutes) appeared to be paraphyletic. With two exceptions, i.e. Thermotoga (Thermotogae) and Fusobacterium (Fusobacteria), which clustered within the Firmicutes, comparative analyses show that at a low taxonomic level, the resolved phylogenetic relationships that were inferred from both the Pgk protein and 16S rRNA gene sequence data are congruent.