Using the Genome Walker procedure, which allows PCR amplification of genomic DNA using a single gene-specific primer and direct automated sequencing methodology, we obtained the nucleotide sequence of the RNA polymerase beta subunit (rpoB) from Bartonella henselae and Bartonella quintana. A phylogenetic tree constructed from these data and other rpoB sequences available in GenBank is, in part, consistent with those previously derived from 16S rRNA gene sequences and confirms the position of Bartonella within the alpha subdivision of Proteobacteria. In fact, this analysis showed that rpoB data are similar to 16S rRNA data for the alpha, beta and gamma subdivisions of Proteobacteria. In contrast, concerning other bacteria included in our study, the topologies of phylogenetic trees were different. Based on the bootstrap values derived from rpoB phylogenic analysis, we believe that this molecule should contribute to better understanding the evolutionary process.