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A PCR assay was developed by using degenerate primers that allow amplification of a 414-bp fragment of DNA from the rickettsia-like organisms Rochalimaea henselae and R. quintana. Internal oligonucleotides were used as hybridization probes, permitting rapid differentiation of these two Rochalimaea species. DNAs from 12 different isolates of R. henselae were amplified with the PCR primers, and the resulting 414-bp PCR product hybridized only with the R. henselae-specific probe. DNAs from four different isolates of R. quintana were amplified and produced a PCR product of the same size that hybridized only with the R. quintana-specific probe. DNAs from isolates of R. elizabethae, R. vinsonii, Bartonella bacilliformis, and Afipia felis failed to amplify the 414-bp fragment in the PCR assay. This two-step assay was applied to DNAs extracted from 16 fresh (unfixed) lymph node biopsy specimens and nine …