This study was undertaken to clarify the in vitro effect of acridine orange (AO) on the cell kinetics of mouse osteosarcoma cells, as well as the mechanism of cell growth inhibition induced by AO. A mouse osteosarcoma cell line (MOS), established from a radiation-induced mouse osteosarcoma, was cultured under exposure to 0.05, 0.5, 5, and 50 micrograms/ml of AO, either continuously or for 10 minutes. The cell kinetic analysis was performed using the following parameters: tumor cell growth by trypan blue exclusion test, mitotic activity, DNA synthetic activity by BrdU labeling and DNA ploidy by cytofluorometry. The results showed that continuous exposure to 5 and 50 micrograms/ml of AO or 10 minute exposure to 50 micrograms/ml of AO quickly killed the tumor cells within 12 hours, whereas continuous exposure to 0.5 microgram/ml of AO or 10 minute exposure to 5 micrograms/ml of AO gradually inhibited tumor cell growth. Under the latter conditions, mitotic activity was rapidly and completely inhibited within 48 hours but DNA synthetic activity was not completely inhibited even after 96 hours. DNA ploidy analysis demonstrated that most of the tumor cells arrested at the S-G2 phase after 12 hours, followed by G2 phase arrest after 24 hours and progressive DNA synthesis to a higher DNA ploidy class after 48 to 96 hours. We therefore concluded that a high concentration of AO has a strong cytocidal effect due to cytotoxicity whilst a moderate concentration of AO induces progressive and synchronous polyploidization by mitotic inhibition without DNA damage in MOS cells. We presume that this in vitro effect on MOS cells may be caused by protein synthetic inhibition after transfer RNA inactivation caused by AO binding.