Rochalimaea quintana was isolated from the blood of a French human immunodeficiency virus-infected patient with bacillary angiomatosis. The isolate showed the typical growth characteristics of Rochalimaea species and was inert when typical biochemical testing was used. The purpose of the present work was to characterize and compare this new isolate with reference strains of R. quintana, Rochalimaea vinsonii, and Rochalimaea henselae by using immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot (immunoblot), restriction fragment length polymorphism-PCR of the citrate synthase gene, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis. SDS-PAGE, Western blot, restriction fragment length polymorphism-PCR with TaqI enzyme, and 16S rRNA gene sequencing could differentiate the three Rochalimaea species and allowed characterization of the French isolate as R. quintana. However, identification of the Rochalimaea isolate to the species level was more easily obtained by immunofluorescence with specific murine antisera. Pulsed-field gel electrophoresis allowed differentiation of the French R. quintana isolate from R. quintana Fuller and may serve as an epidemiological tool.