The Bartonella henselae 17-kDa protein was expressed in a prokaryotic expression system as a histidine-tagged fusion protein and was purified. The target gene was cloned into a recombinant expression construct, pTri-17kd. The expressed protein was purified to near homogeneity by a nickel-agarose column chromatography. Protein recovery was estimated to be 2.9 mg from 100 mL of bacterial culture. The purified 17-kDa protein was recognized by serum from patients infected with B. henselae and Bartonella quintana, suggesting antigenic integrity. The sensitivity and specificity of the IgG enzyme-linked immunosorbent assay (ELISA) relative to immunofluorescent antibody assay testing were 71.1% and 93.0%, respectively. According to the receiver operating characteristic curve analysis, the area under the curve was 0.823. These results indicate that the expressed 17-kDa protein is a suitable source of antigen for development of an antibody-capture ELISA for the detection of antibodies to B. henselae.