The early antibody response in patients with human granulocytic ehrlichiosis (HGE) and in mice infected with the HGE agent was characterized by using sera to probe lysates of HL-60 cells infected with HGE organisms. Sera were obtained from 18 patients with HGE, mostly within the first 6 weeks of clinical infection, and from mice infected with the HGE agent for up to 3 weeks. A 44-kDa antigen was reactive with IgG in all 18 patients, and IgG to 40-, 65-, and 80-kDa antigens was present in 6, 8, and 7 patients, respectively. In addition, IgM to 40-, 44-, 65-, and 80-kDa antigens was found in 9, 5, 4, and 4 subjects. Immunoglobulins to antigens ranging between 95 and 125 kDa were detected less frequently. HGE agent-infected C3H/HeJ mice had an antibody response similar to that in humans. Thus, the 40- and 44-kDa proteins of the HGE agent elicit early strong antibody responses during infection. Characterization of the antigens recognized by antibodies during HGE should aid in diagnosis and understanding of the disease.