Nucleotide base sequence data were obtained for a 940-bp fragment of the citrate synthase-encoding gene (gltA) of representatives of the eight validly described Bartonella species and seven uncharacterized Bartonella strains obtained from small mammals. Complete 16S rRNA gene sequences were also determined for the uncharacterized strains, and these sequences revealed that each strain had a unique sequence which was very similar to the sequences of the previously recognized Bartonella species. A comparison of the gltA sequences of the different Bartonella species revealed that the levels of similarity between sequences were 83.8 to 93.5%, whereas comparisons of sequences obtained from different strains of the same species revealed that the levels of similarity were more than 99.8%. One of the uncharacterized strains had a gltA sequence that matched the sequence of Bartonella elizabethae, three uncharacterized strains had sequences which were more than 99.6% similar to each other (but less than 93.5% similar to any other sequence), and the remaining three uncharacterized strains each exhibited less than 93.5% sequence similarity to other Bartonella species or isolates. Phylogenetic trees were inferred from multiple alignments of both gltA and 16S ribosomal DNA (rDNA) sequences. Whereas the proposed intra-Bartonella architecture of trees inferred from 16S rDNA sequence data by using both distance matrix and parsimony methods had virtually no statistical support, the trees inferred from the gltA sequence data contained four well-supported lineages in the genus. The gltA-derived phylogeny appears to be more useful than the phylogeny derived from 16S rDNA sequence data for investigating the evolutionary relationships of Bartonella species, and the validity of the lineages identified by the gltA analysis is discussed in this paper.