The antibody response to Bartonella henselae has been studied in a number of mammals; however, the human response needs to be further studied. After natural infection, humans have antibody reactivity to a large number of B. henselae proteins. We used a proteomic approach to identify antigenic proteins of B. henselae to determine their capacity to elicit a human antibody response. Comparing patient sera by Western blot analysis demonstrated significant amounts of reactivity to B. henselae. The immunofluorescence assay (IFA)-positive sera identified several protein spots of interest. However, a consistent reactivity to a single spot by all sera was not observed. Three of these spots demonstrated reactivity in 71%, 64%, and 64% of positive sera tested with negligible reactivity to the negative sera. These proteins were identified as GroES, BepA, and GroEL. Most IFA-positive sera demonstrated reactivity to GroES, GroEL, and BepA. The usefulness of these proteins for a clinical serologic assay is discussed.