There are some active transport systems in the cell membrane, such as potassium pump, calcium pump, and proton pump. Although it has been reported that sodium/potassium and sodium/calcium pumps of cell membrane play roles in the intracellular accumulation of anticancer agents, the significance of the active transport channels in accumulation of mitomycin C (MMC), one of the most active agents for non-small cell lung cancer (NSCLC) has been unclear. In this study, we evaluated the role of the potassium pump, calcium pump, and proton pump as determinants of the sensitivity to MMC in vitro by using the selective inhibitors, ouabain, verapamil or AG-2000 (an active metabolite of Lansoplazole), respectively. PC-9 and PC-9/MC4 cell lines which are sensitive and resistant to MMC were used for these experiments. PC-9/MC4 was 9.4-fold more resistant to MMC than PC-9 cells. Relative resistance was not significantly changed by co-incubation with a non-cytotoxic dosage of these inhibitors. From these results, it was revealed that the active transport systems in cell membrane do not play a role in determining the sensitivity to MMC and the acquisition of resistance to MMC in PC-9 cell lines. Intracellular bioactivation may be an important factor to determine sensitivity to MMC in NSCLC cells under aerobic conditions.