Objective: To detect the sodium-independent equilibrative nucleoside transporter (ENT) and sodium-dependent concentrative nucleoside transporter (CNT) on pancreatic cancer cell membrane and make a quantitative assessment of ENT. Methods: Pancreatic cancer cell line (Panc-1) was incubated in the medium containing dipyridamole (100 μmol/L) for 8, 15, 60 and 120 min, respectively. Afterwards, the cells were harvested and lysed with acetonitrile, the fluorescence intensity of dipyridamole in the cell suspension was detected by spectrofluorometer, and the sum of ENTs on a single cell was calculated according to the number of the cells in the parallel suspension. Next, Panc-1 cells were incubated in the medium containing 5-fluorouracil (5-FU) or 5-FU plus dipyridamole for 15, 30, 60, 120 and 240 min, respectively, and the content of 5-FU in the cell was measured by capillary zone electrophoresis; the 5-FU concentration in a single cell was counted according to the number of cells and the single cell volume in the parallel suspension to indirectly determine whether CNT was present on the cell membrane of Panc-1. Results: The fluorescence was detected in Panc-1 after 8 min of incubation in the medium containing dipyridamole with a maximal level of 20.2×10–19 mol at 15 min. The sum of ENTs was determined at 1.25×105 in a single cell according to the dipyridamole content. 5-FU was still transported into the Panc-1 cells after the blockage of ENT with dipyridamole and the maximal level of intracellular 5-FU reached (138.3±9.77) mg/L, which was significantly higher than that of the medium (P=0.011). Conclusion: ENTs on the cell surface can be quantitatively determined through fluorescence intensity detection after their binding with dipyridamole. Whether CNT is present on the cell membrane can be determined by measuring the intracellular 5-FU concentration after the blockage of ENT, and therefore provides a reference for the rational use of chemotherapeutic drugs and improvement of their effect.