Bartonella is a bacterial genus classified in the alpha-Proteobacteria on the basis of 165 rDNA sequence comparison. The highly conserved heat-shock chaperonin protein, GroEL, has proved to be a valuable resolving tool to classify ten Bartonella species. The groEL gene was amplified and sequenced from ten Bartonella isolates: Bartonella alsatica, Bartonella vinsonii subsp. arupensis, Bartonella taylorii, Bartonella tribocorum, Bartonella birtlesii, Bartonella henselae Marseille (URLLY8), B. henselae (90-615), B. henselae (Fizz), B. henselae (CAL-1) and B. henselae (SA-2). Then, phylogenetic relationships were inferred between our isolates and eight other species and subspecies from the comparison of both 16S rDNA and groEL sequences using parsimony, neighbour-joining and maximum-likelihood methods. By using groEL sequences, the first reliable classification of most known Bartonella species and subspecies was established. Four strongly supported subgroups were distinguished: firstly, the two human pathogens B. henselae and Bartonella quintana; secondly, a cluster including four rodent isolates, Bartonella elizabethae, B. tribocorum, Bartonella grahamii and B. taylorii; thirdly, a cluster including the B. vinsonii subspecies (B. vinsonii subsp. vinsonii, arupensis and berkhoffii); and lastly, B. birtlesii and 'Bartonella weissi'. 'Bartonella washoensis', B. alsatica, Bartonella doshiae, Bartonella bacilliformis and Bartonella clarridgeiae did not reliably cluster with any other Bartonella species. In addition, the groEL gene was shown to be useful in subtyping six B. henselae isolates into three variants: Houston, Marseille and Fizz.