Species of the genus Rochalimaea, recently renamed Bartonella, are of a growing medical interest. Bartonella quintana was reported as the cause of trench fever, endocarditis, and bacillary angiomatosis. B. henselae has been implicated in symptoms and infections of human immunodeficiency virus-infected patients, such as fever, endocarditis, and bacillary angiomatosis, and is involved in the etiology of cat scratch disease. Such a wide spectrum of infections makes it necessary to obtain an intraspecies identification tool in order to perform epidemiological studies. B. vinsonii, B. elizabethae, seven isolates of B. quintana, and four isolates of B. henselae were studied by pulsed-field gel electrophoresis (PFGE) after restriction with the infrequently cutting endonucleases NotI, EagI, and SmaI. Specific profiles were obtained for each of the four Bartonella species. Comparison of genomic fingerprints of isolates of the same species showed polymorphism in DNA restriction patterns, and a specific profile was obtained for each isolate. A phylogenetic analysis of the B. quintana isolates was obtained by using the Dice coefficient, UPGMA (unweighted pair-group method of arithmetic averages), and Package Philip programming. Amplification by PCR and subsequent sequencing using an automated laser fluorescent DNA sequencer (Pharmacia) was performed on the intergenic spacer region (ITS) between the 16 and 23S rRNA genes. It was found that each B. henselae isolate had a specific sequence, while the B. quintana isolates fell into only two groups. When endonuclease restriction analysis of the ITS PCR product was done, three enzymes, TaqI, HindIII, and HaeIII, allowed species identification of Bartonella spp. Restriction fragment length polymorphism after PCR amplification of the 16S-23S rRNA gene ITS may be useful for rapid species identification, and PFGE could be an efficient method for isolate identification. Rochalimaea spp. are gram-negative, short-rod bacteria 0.3 to 0.5 by 1.0 to 1.7 m in size. They were included in the family Rickettsiaceae and the tribe Rickettsieae, which was subdivided into three genera: Rickettsia, Coxiella, and Rochalimaea (41). Recently, as a result of 16S rRNA sequencing, the genera Coxiella and Rochalimaea have been removed from the Rickettsiaceae family and the species of the genus Rochalimaea have been renamed Bartonella spp. (4, 39). Bartonella spp. grow on the surface of their eucaryotic host cells but can be grown axenically on blood agar. In the seventh edition of Bergey's manual (41), two species were described: Rochalimaea quintana and R. vinsonii. R. quintana is the etiological agent of trench fever and is transmitted by the human body louse, Pediculus humanus. The disease was first recognized in 1915 in Europe during World War I and afflicted many soldiers. Subsequently, this febrile syndrome occurred in Europe during World War II, as well as in Mexico and in North Africa (36, 37). R. vinsonii has been isolated once from a vole, Microtus pennsylvanicus, trapped on Grosse Isle in the St...