Abstract Background: The ACTS-CC trial is a phase III trial designed to validate non-inferiority of S-1 to UFT/ LV, a standard treatment in Japan as adjuvant chemotherapy for stage III colon cancer and the initial safety report of the trial was reported in ASCO 2011. In biomarker study of ACTS-CC trial, gene expression levels were studied using formalin fixed and paraffin embedded (FFPE) specimens. Methods: Blocks from resected colorectal cancer specimens were obtained under informed consent for this biomarker study from 892 patients enrolled between Apr.2009 and Jan.2010. Gene expression levels of 11 enzymes (TS, DPD, TP, OPRT, FPGS, GGH, DHFR, MTHFR, MTHFD, FOLRA, GART), that correlate to 5-FU and folic acid metabolism, are studied according to Danenberg Tumor ProfileTM method (Shirota, Y. JCO 2001). Correlation coefficients between gene expressions of the 11 enzymes were computed using Spearman rank correlation test (Rs). Results: Gene expression levels were successfully estimated in 521-808 samples (84.0-90.0%, 89.4% in median). Highest Rs was observed between DPD and TP (0.68. 95% confidence interval; 0.64-0.71). Classifying Rs value into 3 groups (positive (0.5<), week (0.31-0.50) and negative (0.3 or less)), TS and OPRT had positive or week correlation with 4 of 7 metabolic enzymes of folic acid (DHFR, MTHFR, MTHFD, and GART). Conclusions: This is the first and largest gene expression analysis for metabolic enzymes of 5-FU and folic acid. Positive correlation was observed between gene expression level of DPD and TP, which are DNA catabolic enzymes. Gene expression level of TS and OPRT, which are DNA synthetic enzymes, had correlation with metabolic enzymes of folic acid. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4516. doi:1538-7445.AM2012-4516