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Monoclonal antibodies were raised against isolated spindles of CHO (Chinese hamster ovary) cells to probe for molecular components specific to the mitotic apparatus. One of the antibodies, CHO1, recognized an antigen localized to the midbody during mitosis. Immunofluorescence staining of metaphase cells showed that although the total spindle area was labeled faintly, the antigen corresponding to CHO1 was preferentially localized in the equatorial region of the spindle. With the progression of mitosis, the antigen was further organized into discrete short lines along the spindle axis, and eventually condensed into a bright fluorescent dot at the midzone of the intercellular bridge between two daughter cells. Parallel immunostaining of tubulin showed that the CHO1-stained area corresponded to the dark region where microtubules are entrapped by the amorphous dense matrix components and possibly blocked from binding to tubulin antibody. Immunoblot analysis indicated that CHO1 recognized two polypeptides of mol wt 95,000 and 105,000. The immunoreaction was always stronger in preparations of isolated midbodies than in mitotic spindle fractions. The protein doublet was retained in the particulate matrix fraction after Sarkosyl extraction (Mullins, J. M., and J. R. McIntosh. 1982. J. Cell Biol. 94:654-661), suggesting that CHO1 antigen is indeed a component of the dense matrix. In addition to the equatorial region of spindles and midbodies, CHO1 also stained interphase centrosomes, and nuclei in a speckled pattern that was cell cycle-dependent. Thus, the midbody appears to share either common molecular component(s) or a similar epitope with interphase centrosomes and nuclei.