The bacterial genus Bartonella (Rochalimaea) includes emerging human pathogens with five recognized species. These are fastidious gram-negative bacteria, exhibiting few phenotypic characteristics and whose identification relies upon serotyping, cellular fatty acid analysis, and molecular typing. Most of the isolates have been recovered from the blood of patients, and three of the four pathogenic Bartonella species are associated with infectious endocarditis. We performed PCR-restriction fragment length polymorphism (RFLP) analysis of the blood culture bottle supernatant for the routine identification of Bartonella species among fastidious gram-negative bacteria. The amplification of the citrate-synthase gene with primers previously reported (R. L. Regnery, C. L. Spruill, and B. D. Plikaytis, J. Bacteriol. 173:1576-1589, 1991) yielded a 379-bp product from Bartonella species and a 382-bp product for Capnocytophaga ochracea but no product from any of the other 15 genotypically or phenotypically related species tested. We determined the sequences of the citrate-synthase gene-amplified products for Bartonella species and C. ochracea in order to predict the optimal restriction enzyme to be used in RFLP analysis. TaqI and AciI allowed identification of Bartonella species and C. ochracea. We propose that acridine orange and Gram staining, followed by PCR-RFLP analysis of the blood bottle supernatant, be included in the examination of blood samples from patients with suspected infectious endocarditis.