Bartonella koehlerae, a recently described feline Bartonella species, was isolated from two naturally infected cats in northern California. We experimentally infected domestic cats with B. koehlerae to establish the microbiological and immunological characteristics of this infection in cats and to compare it to infections with those caused by B. henselae and B. clarridgeiae. Four cats were inoculated intradermally with B. koehlerae (8.6 x 10(7) to 3.84 x 10(8) CFU/ml). None of the cats presented any obvious clinical signs, but all cats developed bacteremia, which peaked at 3.36 x 10(4) to 1.44 x 10(6) CFU/ml of blood between day 14 and day 36 postinoculation. B. koehlerae-inoculated cats had a bacteremia duration (mean, 74 days) shorter than did cats inoculated with B. clarridgeiae (mean, 324 days) (P = 0.03). None of the four cats inoculated with B. koehlerae had bacteremia relapse. As shown by enzyme-linked immunosorbent assay (ELISA) using B. koehlerae outer membrane protein (OMP) antigens, the four cats developed a species-specific antibody response, and ELISA testing using other feline Bartonella OMP antigens showed statistically lower optical density values. All four cats developed similar antibody reactivity patterns to B. koehlerae OMP antigens as seen by Western blotting, each with at least 20 seroreactive protein bands. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protein profile differences were observed for both whole-cell lysate and OMPs from B. koehlerae, compared with B. henselae and B. clarridgeiae. B. koehlerae was more closely related to B. henselae than to B. clarridgeiae by protein profile, and this relatedness was also confirmed by analysis of the genomic DNA profiles by pulsed-field gel electrophoresis.