Thymidylate synthase (TS) is a key enzyme in the synthesis of DNA and the target enzyme of 5-fluorouracil, the most widely used chemotherapeutic agent for colon cancer. The expression of TS messenger RNA or protein reportedly predicts overall survival for colon cancer patients and correlates with resistance to 5-fluorouracil. Because the gene encoding TS is thought to be one of the target genes bound to and regulated by the E2F1 transcription factor, it was of interest to measure TS and E2F1 gene expressions in primary colon cancer specimens. We found that TS expression correlates closely with E2F1 expression in colon cancer specimens. Moreover, this correlation was observed in all tumors, regardless of clinical stage. These results suggest that the ability of a tumor to increase TS expression may be due to an over-expression of E2F1, and they provide new insights into the molecular mechanisms underlying the regulation of TS expression in colon cancers.