BACKGROUND The fastidious nature of Bartonella henselae is such that demonstration of clinical infection relies mainly on serological or molecular biological methods. Only five isolates have been obtained from patients with cat-scratch disease (CSD) and none from endocarditis. METHODS We isolated B henselae from a CSD patient and, for the first time, from a patient with endocarditis. The isolates were characterised on the basis of morphology, biochemistry, cell-wall fatty-acid analysis, PCR-restriction fragment length polymorphism analysis of the 16-23S intragenic spacer region, and 16S rRNA gene sequences. Characterisation of these isolates indicated them to belong to a new serogroup, which we have called "Marseille", and to a new genotype based on the 16S rRNA gene sequence. The new variant was incorporated into an immuno-fluorescence antibody test (IFAT), which was used to reassess serum samples from 113 CSD patients who were seronegative with the conventional IFAT. RESULTS 18 (16%) of these apparently seronegative patients yielded significantly raised titres. 20 CSD patients who were seropositive as judged by the conventional IFAT remained seropositive with the new IFAT. CONCLUSIONS Antigenic variability within the species is one possible reason for inconsistent results in the serological diagnosis of CSD.