Interaction between 5-fluorouracil (5-FU) and FdUMP[10], a novel pro-drug formulation of the thymidylate synthase (TS) inhibitory nucleotide 5-fluoro-2'-deoxyuridine-5'-O-monophosphate (FdUMP), was investigated to evaluate the feasibility of using these two forms of fluorinated pyrimidine in combination chemotherapy regimens. 5-FU and FdUMP[10] are expected to differ in their relative intracellular distribution of active metabolites, and their combined administration may result in either a positive or a negative interactive effect. The dose-response behaviors of 5-FU and FdUMP[10] toward H630 and H630-10 (human colorectal tumor) cells were first investigated separately. Effects on cell viability were measured using an assay for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), while cytotoxicity and apoptosis were investigated using clonogenic and TUNEL assays, respectively. Exposure of H630 cells to concentrations of FdUMP[10] insufficient to inhibit cell proliferation as a single agent markedly increased the cytotoxicity of 5-FU. The results indicate that 5-FU and FdUMP[10] interact in a positive manner, and that combining these two forms of fluorinated pyrimidine may be clinically beneficial.