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Multidrug resistance (MDR) is a major problem in cancer chemotherapy. It was previously reported that a red ginseng saponin, 20(S)-ginsenoside Rg₃ could modulate MDR in vitro and extend the survival of mice implanted with ADR-resistant murine leukemia P388 cells. This study examined the cytotoxicity of Rg₃ on normal and transformed cells, along with its effect on the membrane fluidity. The cytotoxicity study revealed that 120 μM of Rg₃ was cytotoxic against a multidrug-resistant human fibroblast carcinoma cell line, KB V20C, but not against normal WI 38 cells in vitro. Flow cytometric analysis using rhodamine 123 as the artificial substrate showed that Rg₃ promoted the accumulation of rhodamine 123 in ADR-resistant murine leukemia P388 cells in vivo. Fluorescence polarization studies using the hydrophilic fluorescent probe, DPH, and hydrophobic probe, TMA-DPH, showed that 20 μM Rg₃ …