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: The purpose of this study is to investigate heterogenous cell populations in cultured rat periodontal ligament (PDL) cells and to isolate different cell populations from PDL cells. Outgrowing rat PDL cells obtained from the mandibular incisors were cultured for 24 d ays and alteration of cells was observed . A f ew foci appeared in the culture on day 8 and they enlarged . The cells in the foci expressed osteocalcin (Osc) by immunohistochemistry , and they formed mineralized nodules on day 24 , but the cells from the surrounding area did not . Osc production from the cells by immunoassay was closely related to enl argement of the foci. The cells cultured on thin membranes were isolated on day 18 b y inserting fine-point pasteur pipets into the center of each focus and the surrounding area under the dissecting microscope . Most of the cells isolated from the focus expressed Osc protein and mRNA , while the cells isolated from the surounding area expressed neither Osc protein nor mRNA . These Osc-expressing or non-expressing cell populations would be useful to investigate the precise biological function and inte raction between different cell populations of PDL cells.