The cell proliferation-associated nuclear antigen recognized by the Ki-67 monoclonal antibody was detected immunohistochemically in cells from skeletal tissues of humans, sheep and rabbits. In each case the distribution of Ki-67-positive cells was identical and consistent with the known distribution of dividing cells in growing long bones. The antigen recognized by Ki-67 is detectable only after fixation in cold acetone. In the 5-day-old rabbit, labelling with tritiated thymidine ( 3HTdR) in vivo, followed subsequently by a combination of Ki-67 immunohistochemistry and autoradiography for 3HTdR, demonstrated that, in addition to the expression of Ki-67 by the majority of 3HTdR labelled cells, other cells associated with proliferating populations also expressed the Ki-67 antigen. This suggests that the Ki-67 antibody identifies cycling cells in the skeleton in S-phase, as well as in other phases of the cell cycle. In Ki-67 reactive species, this method is a potentially useful tool for the study of cell proliferation in skeletal tissues in vivo and in vitro, having the advantage of not requiring prior labelling with DNA precursors.