Bartonella bacilliformis is the etiologic agent of Oroya fever in humans. Flagellum-mediated motility has been postulated as a major virulence factor for invasion of host cells. To address this hypothesis, we purified and characterized flagella from strain KC584 and then assessed their role in human erythrocyte association and invasion. Electron microscopy of the flagellar preparation showed a high concentration of filaments with a mean wavelength of 800 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, and KBr density gradient centrifugation indicated that the flagellar filament is composed of a polypeptide of 42 kDa. The flagellin is partially (ca. 50%) resistant to treatment with trypsin. The first 17 amino acid residues of the N terminus of the mature flagellin protein are GAAILTNDNAMDALQDL and show approximately 46% sequence identity to the residues of the N termini of two Caulobacter crescentus flagellin proteins. A monospecific polyclonal antibodies to the flagellin protein was generated, and its specificity was verified by both immunoblot and immunogold analyses. Human erythrocyte invasion assays performed with bartonellae exposed to the antiflagellin antiserum showed a significant decrease in bacterial association with and invasion of human erythrocytes in comparison with that in bartonellae exposed to preimmune rabbit serum or phosphate-buffered saline (PBS) controls. These results suggest that flagella are an important component in the invasiveness of B. bacilliformis.