There have been many reports concerning the intracellular binding sites of acridine orange (AO), although the actual localization of AO in living cells remains controversial. This study was undertaken to clarify the intracellular localization of AO in living mouse osteosarcoma cells by cytochemical staining. A mouse osteosarcoma cell line (MOS) was cultured and continuously exposed to 0.5 microgram/ml of AO. The intracellular localization and stainability of AO the living tumor cells was morphologically detected by a high resolution fluorescence microscope. To detect the intracellular microstructure, cytochemical staining with rhodamin 123 for mitochondria, acid phosphatase for lysosome, Sudan-black for fat vesicle and toluidine blue for glucosaminoglycan were performed using fixed cells. The results showed that both the nucleus and cytoplasm of tumor cells at 10 minutes after exposure to 0.5 microgram/ml of AO emitted green fluorescence, which was especially intense in the nucleolus, but not brilliant in the nucleus and was granular orange to red fluorescence in the perinuclear particles. This stainability of AO was different from that of rhodamin 123, Sudan-black or toluidine blue, but similar to that of acid phosphatase. Based on these results, we conclude that the green fluorescence may have derived from AO binding to double stranded RNA, not to DNA, and that orange fluorescence may have derived from aggregated AO binding to lysosome.