Intracellular reduced folate exists as a “pool” of more than 6 interconvertable forms. One of these forms, 5,10 methylenetetrahydrofolic acid (CH2THF), is the key one-carbon donor and reduced folate substrate for thymidylate synthase (TS). This pathway has been an important target for chemotherapy as it provides one of the necessary nucleotide substrates for DNA synthesis. The fluoropyrimidine 5-fluorouracil (5-FU) exerts its main cytotoxic activity through TS inhibition. Leucovorin (5-formyltetrahydrofolate; LV) has been used to increase the intracellular reduced folate pools and enhance TS inhibition. However, it must be metabolized within the cell through multiple intracellular enzymatic steps to form CH2THF. CoFactor (USAN fotrexorin calcium, (dl)-5,10,-methylenepteroyl-monoglutamate calcium salt) is a reduced folate that potentiates 5-FU cytotoxicity. According to early clinical trials, when 5-FU is modulated by CoFactor instead of LV, there is greater anti-tumor activity and less toxicity. This review presents the emerging role of CoFactor in colorectal and nongastrointestinal malignancies.