No control cell line was available for previous RNA interference studies on reversal of multidrug resistance (MDR) in colon cancer cells. Here, human COLO 320DM, with HT-29 as the control, colon cancer cell lines were used to investigate the reversal of MDR1/P-gp-dependent MDR by siRNA (#4123 and #4029 MDR1 siRNAs) targeting to MDR1 mRNA. Both siRNAs inhibited expression of MDR1 and P-gp in COLO 320DM. The minimum inhibition concentrations were 5 nmol/l of #4123 and 25 nmol/l of #4029. #4123 MDR1 siRNA took effect in 4, 5 and 6 days at doses of 5, 25 and 100 nmol/l, respectively. Increased cytotoxicity of the antitumor drugs adriamycin and vincristine with increased intracellular adriamycin accumulation accompanied inhibition of MDR1 mRNA and P-gp expression. No such effects were found in the HT-29 control. MDR1 siRNAs specifically reversed the MDR of colon cancer cells demonstrating a possible new approach for treating MDR1/P-gp-dependent multidrug resistance.