The purpose of this study was to determine simple genetic factors helpful to tailor 5-FU administration and determine strategy in first-line chemotherapy of advanced colorectal cancer. In 76 patients initially treated by 5-FU, thymidylate synthase, dihydropyrimidine dehydrogenase and methylene tetrahydrofolate reductase germinal polymorphisms, dihydrouracil/uracil plasma ratio and 5-FU plasma clearance were investigated and correlated for tolerance (10.5% grade 3 and 4 toxicity) and efficacy (32.9% objective response rate and 20 months median overall survival time). Toxicity was linked to performance status >2 (P=0.004), low UH2/U ratio, 2846 A>T, IVS 14+1G>A for DPD (P=0.031), and homozygoty C/C for MTHFR 1298 A>C (P=0.0018). The overall survival of the patients with a 3R/3R TS genotype associated with C/C for 677 C>T or A/A for 1298 A>C was statistically shorter (log-rank test P=0.0065). Genetic factors permit the tailoring of 5-FU treatment. They should occupy center stage in future clinical trials for specifically designing treatment for patients with a given biologic feature.