755 Background: 5-fluorouracil (5-FU) chemotherapy is associated with severe and unpredictable toxicity in a significant proportion of patients.We hypothesized that susceptibility to 5-FU toxicity might be related to individual differences in components of reduced folate metabolism affecting intracellular 5,10-MTHF levels, detectable in blood prior to treatment. Methods: A prospective cohort of chemo-naive colorectal cancer patients planned to receive IV 5-FU and folinic acid for five consecutive days every four weeks were studied. Baseline clinical and laboratory data were collected prior to treatment. Biochemical data associated with folate metabolism was also collected and not revealed to treating physicians. The primary endpoint was occurrence of > grade 3 toxicity and/or toxicity mandating dose delay or reduction. Results: Of 78 eligible patients studied, 68% experienced > grade 3 toxicity, 69% had schedule modification, and 81% had either or both. Multivariable analyses identified only a higher pre-treatment serum folate level as an independent predictor of toxicity > grade 3 and/or mandating schedule modification (p = 0.016). An increasing toxicity trend was observed amongst folate-stratified patient cohorts, with an odds ratio of 2.87 (p = 0.09) comparing the highest and lowest quartiles. Concurrently, overall survival and relapse-free rates also increased with pretreatment folate levels in the adjuvant cohort with log rank values of 3.60 (p = 0.06) and 7.20 (p = 0.007) between the highest quartile and the lower quartiles, respectively. Conclusions: The incidence of severe toxicity with this schedule of 5-FU and folinic acid was high and positively correlated with the pretreatment serum folate level. Interestingly, a concurrent increase in overall and relapse free survival is observed with increasing serum folate levels. These results suggest that an optimal pretreatment folate level exists that balances chemotherapy treatment efficacy and toxicity.