Two bacterial strains, one isolated from the blood of a dog with valvular endocarditis and one isolated from the blood of a healthy dog, were similar to Bartonella species, as determined by a number of phenotypic criteria, including growth characteristics, biochemical reactions, and cell wall fatty acid composition. The results of 16S rRNA gene sequence similarity studies confirmed that these strains are closely related and belong in the genus Bartonella and that Bartonella vinsonii is their closest relative (the 16S rRNA of isolate 93-C01T [T = type strain] was 99.37% identical to the 16S rRNA of the type strain of B. vinsonii, the 16S rRNA of isolate G7464 was 99.61% identical to the 16S rRNA of the type strain, and the 16S rRNAs of the dog isolates were 99.77% identical to each other). The 16S rRNAs of both strains contained a 12-base insertion that was not present in the 16S rRNA of the type strain of any Bartonella species. DNA relatedness tests revealed that these strains were related at the species level to the type strain of B. vinsonii. They were, however, significantly more closely related to each other than to B. vinsonii. On the basis of their unique 16S rRNA sequence insertion, their preferentially high level of relatedness, and their similar origins (dogs), we believe that strains 93-C01(T) and G7464 should be placed in a separate subspecies of B. vinsonii, for which we propose the name B. vinsonii subsp. berkhoffii subsp. nov. The type strain of B. vinsonii subsp. berkhoffii is strain 93-C01 (= ATCC 51672). The description of B. vinsonii is emended to accommodate the new subspecies, and B. vinsonii subsp. vinsonii is described.