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Vegetative valvular endocarditis involving the aortic and, to a lesser extent, mitral valves was diagnosed echocardiographically in a 3-year-old spayed female Labrador retriever. Historically, the dog had been treated with tetracycline hydrochloride and prednisolone for positive seroreactivity to Ehrlichia canis and antinuclear antigens. Although three aerobic and anaerobic blood cultures failed to grow bacteria, blood cultured simultaneously by the lysis centrifugation technique grew a fastidious, gram-negative organism. Despite an initial therapeutic response, the owner elected euthanasia 17 days later. Necropsy confirmed aortic and mitral valvular endocarditis. Bacteria phenotypically similar to Bartonella species were visualized in the heart valve by light and electron microscopy, and Bartonella DNA from a frozen heart valve was amplified by PCR. Subsequent phenotypic and genotypic characterization of the isolate, including biochemical testing, cellular fatty acid analysis, DNA hybridization, and sequencing of the 16S rRNA gene indicated that this organism, which can induce endocarditis in dogs, is a novel Bartonella subspecies containing an insertion sequence unique among currently recognized Bartonella species. The name Bartonella vinsonii subsp. berkoffii subsp. nov. will be proposed for this organism.