Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium that replicates slowly in the host cytoplasm. To investigate the changes of antigen expression during six days of in vitro growth, the reactivity of various monoclonal antibodies (MAbs) was examined in time course. Using immunofluorescence staining, some antigens were shown to be differentially expressed in contrast to 56-kDa protein that was produced at a constant level throughout culture. Three MAbs (NT1, M716B and M716G) revealed antigens, appearing only at 3 days after infection. MAb NT19 recognized an antigen that appeared mainly at the late stage of infection whereas two MAbs (M686-13 and M686-20) demonstrated antigens, being expressed at the early infection stage and showing distinct morphology under immunofluorescence staining. In addition, when cells were infected and then treated with chloramphenicol, granular cytoplasmic bodies were detected and the bacterial development was inhibited. These results show that O. tsutsugamushi changes the antigenic expression to adapt to and replicate in host cells.