The new B. koehlerae subspecies shows a unique PCR-RFLP pattern for the citrate synthase gene (gltA) and a unique PFGE pattern that differs from those of B. henselae and B. koehlerae. The type strain, L-42-94, recovered from the blood of a mountain lion (Puma concolor) during a prevalence study of Bartonella infections in wild cats in northern California, has been deposited at the ATCC, USA (ATCC BAA-2635; http://www.atcc.org/) and at the Collection of Institut Pasteur (CIP), Paris, France. Here, CS is linked to bartonellosis.