N5, N10-Methylenetetrahydrofolate (MTHF) is a cofactor, which is essential for the reductive methylation of 2’-deoxyuridine-5’-monophosphate (dUMP) RO 2’-deoxythymidine-5’-monophosphate (dTMP) catalyzed by thymidylate synthase (TSase). A recent in vitro study conducted in our group has provided evidence that human cancer cell lines with over-expressed TSase protein and membrane-bound folate receptors exhibit three-to ten-fold higher uptake of MTHF, when compared with the normal cells from the same organs. We envisioned that by inhibiting the intracellular synthesis of MTHF, the cells might depend more on the supply of extracellular MTHF, thus enhancing the uptake of MTHF. This hypothesis was tested by treating head & Neck (Cal 27) cancer cell line with a potent inhibitor of dihydrofolate reductase (DHFR) i.e., methotrexate (MTX), and observing the uptake of MTHF by these cells. In this research, Cal27 cells were first sequentially adapted to folate free medium, and the pre-treated with MTX (10 Nm) for different time points, followed by treatment with a single dose of 20 µM MTHF containing tracer amount of [14C]-MTHF for 1h. After removal of tracer containing medium, the cells were detached and centrifuged to a pelt, which was lysed in scintillation fluid and counted by liquid scintillation counter (LSC). The result indicated that Cal27 cells accumulate more radio-tracer when incubated with MTX in folate free medium as compared to those that were grown in normal medium. These results will provide essential information for the development of MTHF as a potential Positron Emission Tomography (PET) probe in clinical diagnosis, management and treatment of human cancer.