We amplified by polymerase chain reaction (PCR) and sequenced using an automated laser fluorescent DNA sequencer (Pharmacia) the intergenic spacer region (ITS) between the 16S and 23S rRNAs of the four species of Rochalimaea which were recently renamed Bartonella sp. We obtained DNA fragments of 1211, 1262, 1258 and 1529 bp for the reference species of B. quintana, B. henselae, B. vinsonii and B. elizabethae, respectively. The ITS of the four species are longer than previously reported in prokaryotes and contained the genes encoding isoleucine-tRNA (tRNA(Ile)) and alanine-tRNA (tRNA(Ala)). The sequences of the tRNA(Ala) genes are identical for the four Bartonella species, but the tRNA(Ile) gene sequence of B. quintana presents one mutation in comparison with the other species.