The relationship of thymidylate synthase (TS) and methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms on 5-fluorouracil (FU) sensitivity was tested on 19 human cancer cell lines (head and neck, breast, digestive tract) in the absence and presence of folinic acid (FA) supplementation. Thymidylate synthase polymorphisms in the 5 0 promoter region (double or triple tandem repeats) and 3 0 untranslated region (6-bp deletion) were analysed by PCR. The C677T and A1298C MTHFR polymorphisms were determined by melting curve analyses (LightCycler). Thymidylate synthase activity and intracellular concentration of the reduced folate 5-10 methylenetetrahydrofolate (CH 2 FH 4 ) were measured (biochemical assays). Thymidylate synthase activity was significantly different according to 5 0 TS genotype, heterozygous cell lines (2R/3R) exhibiting higher TS activities than homozygous ones (P ¼ 0.05). However, whether in the absence or presence of FA, FU sensitivity was not statistically associated with either 5 0 or 3 0 TS polymorphism. Basal CH 2 FH 4 cellular concentrations were lowest in C677T homozygous wild-type (wt) (C/C) cell lines. FU sensitivity was not linked to C677T polymorphism. In contrast, there was a marked trend for a greater FU efficacy in mutated A1298C variants (C/C þ A/C) as compared to wt homozygous cell lines (A/A) (P ¼ 0.055 and 0.085 without and with FA supplementation, respectively). These results suggest for the first time a potential role of A1298C MTHFR polymorphism on fluoropyrimidine sensitivity.