A large number of Bartonella species and genetic variants were compared for their ability to cause bacteremia in different rodent species: the cotton rat (Sigmodon hispidus), white-footed mouse (Peromyscus leucopus), BALB/c mouse and Wistar rat. Experimental data supported field observations that host specificity can occur among certain Bartonella species and rodent species. Bacteremia could only be readily produced in cotton rats or white-footed mice if the strains used for inoculation were originally obtained from the same species or from a phylogenetically close species. A few Bartonella colonies could be observed in the blood of some BALB/c mice by 7 days after inoculation, but no evidence of the persistence of the infection was found. Host specificity suggests the possibility of a long co-speciation of Bartonella species with their rodent hosts. Host-parasite relationships measured by the duration and level of bacteremia and the minimal infectious dose may serve as additional criteria for classification of Bartonella isolates obtained from natural environments.