Cytotoxic effect of progesterone on the neoplastic cells was investigated using POS cells, a cell line established from a spontaneous osteosarcoma in a dog. The 20 alpha-HSD activity of POS cells was 0.85 +/- 0.26 NADPH nmol/min mg protein, demonstrating that a considerable activity was present in this tumor cell. When progesterone was added to the medium and cultured for 48 hr, progesterone dose-dependently inhibited the cell growth, showing that progesterone was cytotoxic on this osteosarcoma cells in vitro. In order to assure the role of 20 alpha-HSD on the cell growth, various concentrations of four types of steroid derivatives, STZ 20, 23, 25, and 26, potent inhibitors of 20 alpha-HSD, were added to the medium with 0.1 microM progesterone. Then, the POS cell growth was more strongly inhibited, which may suggest that the cytotoxicity of progesterone was enhanced by inactivation of 20 alpha-HSD by STZs. From these results, POS osteosarcoma cells have 20 alpha-HSD, which might play an important role in tumor cell growth against the cell toxicity of progesterone.