Intracellular bacteria often change the expression of their genes in order to adapt to new environmental conditions. Here we describe a monoclonal antibody (MAb) that reacts exclusively against intracellular Orientia tsutsugamushi. Although MAb applied to the 56-kDa protein, a major outer membrane protein, reacted against a large number of bacteria that had attached to host cells at the early stage of infection, M686-13 reacted against only a minor portion of the attached bacteria. In the later stage of the intracellular growth cycle, both antibodies showed identical staining patterns by double immunofluorescent staining. These results suggest that M686-13 reacted to an epitope or a protein that had probably been expressed during the intracellular growth cycle and rapidly diluted or degraded upon release into the extracellular environment. Although its molecular characteristics remain unknown, the reactive antigen may prove to be a novel developmental antigen and this MAb could be used as reagent for the staining of viable O. tsutsugamushi.