It is difficult to distinguish infections with different Bartonella species using commercially available immunofluorescence (indirect immunofluorescent antibody [IFA]) assay kits. To identify appropriate proteins for serodiagnosis of Bartonella quintana infections, we investigated the antigenicity of B. quintana proteins using sera from homeless people with high B. quintana IgG titers in IFA assay. These sera reacted strongly to an outer membrane protein, hemin-binding protein D (HbpD). Further, serum from an endocarditis patient infected with B. quintana reacted to HbpB and HbpD. To locate the antigenic sites within the proteins, we generated deletion mutants of HbpB and HbpD. Amino acid residues 89 to 220 of HbpB and 151 to 200 of HbpD were identified as the minimum regions required for recognition by these sera. Several oligopeptides comprising parts of the minimum regions of HbpB and HbpD were synthesized, and their immunoreactivity with the above-mentioned sera was tested by enzyme-linked immunosorbent assay (ELISA). Serum from the endocarditis patient reacted similarly to synthetic peptides HbpB2 (amino acid residues 144 to 173 of HbpB) and HbpD3 (151 to 200 residues of HbpD), while sera from the other subjects reacted to HbpD3. These results indicate that synthetic peptides HbpB2 and HbpD3 might be suitable for developing serological tools for differential diagnosis of B. quintana infections from other Bartonella infections.