Bartonella bacilliformis and Bartonella henselae, the respective agents of Oroya fever and cat-scratch disease in humans, are known to produce bacteriophage-like particles (BLPs) that package 14 kbp segments of the host chromosome. Data from this study suggest that other Bartonella species including Bartonella quintana, Bartonella doshiae and Bartonella grahamii also contain similar BLPs, as evidenced by the presence of a 14 kbp extrachromosomal DNA element in their genomes, whereas Bartonella elizabethae and Bartonella clarridgeiae do not. A purification scheme utilizing chloroform, DNase I and centrifugation was devised to isolate BLPs from B. bacilliformis. Intact BLPs were observed by transmission electron microscopy and were round to icosahedral in shape and approximately 80 nm in diameter. RFLP and Southern blot analysis of BLP DNA from B. bacilliformis suggest that packaging, while non-selective, is less than the near-random packaging previously reported for the B. henselae phage. Data also suggest that the linear, double-stranded BLP DNA molecules have blunt ends with noncovalently closed termini. Packaging of the BLP DNA molecules into a protein coat appears to be closely related to nucleic acid synthesis, as unpackaged phage DNA is not detectable within the host cell. SDS-PAGE analysis of purified BLPs from B. bacilliformis showed three major proteins with apparent molecular masses of 32, 34 and 36 kDa; values that closely correspond to proteins found in B. henselae BLPs. Western blot analysis performed with patient convalescent serum showed that BLP proteins are slightly immunogenic in humans. To determine if BLPs contribute to horizontal gene transfer, mutants of B. bacilliformis were generated by allelic exchange with an internal fragment of the 16S-23S rDNA intergenic spacer region and a suicide vector construct, termed pKB1. BLPs from one of the resultant strains were able to package the mutagenized region containing the kanamycin-resistance cassette; however, numerous approaches and attempts at intraspecies transduction using these BLPs were unsuccessful.