This study aimed to prospectively evaluate the potential influence of folate status on the toxicity induced by 5-fluorouracil (5-FU)-based chemotherapy in patients with gastrointestinal tumors. 105 patients with colorectal, pancreatic or gastric cancer were entered into the study. Treatment regimens consisted of bolus 5-FU/leucovorin or infusional 5-FU combined with cisplatin. Baseline homocysteine, vitamin B(12) and folic acid serum levels were determined in all patients. Univariate and multivariate logistic regression models were used to identify predictive factors for toxicity. Univariate analysis showed a significant association between older age, low BSA, gastric/pancreatic cancer and treatment with 5-FU/cisplatin and the incidence of grade 3-4 hematological toxicity, and between female sex, low BSA and gastric/pancreatic cancer and the incidence of severe non-hematological toxicity. Variables that retained independent prognostic value in the multivariate model were tumor type, chemotherapy schedule and BSA for both hematological and non-hematological toxicities. Baseline homocysteine, vitamin B(12) or folate status were not significant predictors of any kind of toxicity either according to univariate or multivariate analysis. This study failed to demonstrate a significant association between a patient s nutritional folate status and the toxicity induced by fluoropyrimidine-based chemotherapy in a cohort of patients with various gastrointestinal malignancies.