OBJECTIVE Anti-cancer effect of 5-Fluorouracil (5-FU) is mediated mainly by inhibition of the thymidylate synthase (TS), while dihydropyrimidine dehydrogenase (DPD) is an initial and a rate-limiting catabolic enzyme of 5-FU. In this study, the mRNA expression profiles of TS, DPD, p53 and c-myc were investigated in mouse liver. METHODS A total of 24 male B6D2F1 mice were involved in this study. All the mice were synchronized with an alternation of 12 h of light (L) and 12 h of darkness (D) (LD12:12) for 4 weeks. Body temperature and rest-activity were monitored with an intra-peritoneal sensor. All the mice were sacrificed at 3, 7, 11, 15, 19, 23 HALO (hours after light onset) respectively and liver samples were obtained and immediately frozen in liquid nitrogen. Total RNA was extracted from the frozen liver samples and one-step real-time quantitive RT-PCR was performed using LightCycler - RNA Amplification Kit SYBR Green I system. RESULTS Both body temperature and rest-activity displayed similarly rhythmic patterns with peak times located in darkness, while the trough time was located in the light span. DPD showed a circadian expression in mRNA level with a peak at about 16 HALO (P=0.0012). TS showed a trend for a circadian rhythm, with a peak during light (P=0.079). Neither c-myc nor p53 displayed significant circadian rhythm. CONCLUSIONS The 24-h patterns in DPD and TS expression were approximately 12 h out of phase, supporting a coordinated regulation of both transcriptional pathways relevant for 5-FU chronopharmacology.