Approximately one-third of all cases of colorectal carcinoma present in an advanced and, therefore, incurable stage. For these patients, the development of new chemotherapeutic strategies is of central importance. Biochemical modulation of 5-fluorouracil (5-FU) has resulted in approximately a two-fold increase in activity of 5-FU. Recent preclinical investigations suggest that interferon can also modulate the activity of 5-FU and may result in enhanced response rates in patients. One of the critical mechanisms of resistance to 5-FU appears to be the acute induction in thymidylate synthase (TS) levels following therapy with inhibitors of this enzyme. This mechanism is based on a novel autoregulatory feedback pathway wherein the TS protein regulates its own translational efficiency. Regulatory function of the enzyme is dependent on its state of occupancy by either the physiologic ligands or inhibitors, including fluoropyrimidines and antifolates. Ongoing efforts are directed towards utilizing knowledge of this protein/messenger RNA interaction for therapeutic benefit. Given the importance of TS, our laboratory has developed antibodies capable of quantitating the levels of this enzyme in fresh or paraffin-embedded tissues. Preliminary investigations suggest that the level of TS has prognostic importance in patients with rectal carcinoma and may be used to predict responsiveness to fluoropyrimidine agents. Novel strategies utilizing dual modulation of 5-FU with leucovorin and interferon are under investigation in both the advanced and adjuvant disease settings. Emerging mechanistic concepts regarding TS, along with the development of new, more potent inhibitors will hopefully result in future therapeutic gains.