Introduction: 5-Fluorouracil (5-FU) is the basis of most combination chemotherapies for gastrointestinal tumors. It is generally well tolerated, but side-effects might require dose-adjustment. As adverse events are not specific to the 5-FU component of the chemotherapy-combination, i.e. neutropenia, diarrhea or cardiotoxicity, the knowledge of 5-FU serum levels might help to attribute these side effects to the 5-FU compound. The optimal concentration-range (AUC, area under the curve) has been described to be within 20-25 mgh/l. The aim of this study was to analyse the intra- and interindividual variability of 5-FU AUC-levels in patients with 5-FU infusion therapy. Methods: 230 blood samples were obtained from 31 different gastrointestinal cancer patients (esophagus (8), stomach (10), ileum (1), colorectum (12)) treated with 5-FU-infusional regimes, based on a 24- or 48-hour AIO treatment-schedule. 5-FU plasma concentrations were measured using an immunolinked Elisa assay (Saladax 5-FU PCMTM). Intra- and interindividual differences were analysed before (0 h; n = 115), at 2 - 3 hours after the start of infusional 5-FU treatment (n = 19) (early sampling) and towards the end of the infusion (n = 96) (late sampling). Results: Early blood sampling resulted in low 5-FU plasma concentrations (541 ± 127 g/ml) due to saline prefilling (2 - 3 ml) of the Baxter pump. Blood sampling at the later time-point resulted in reproducible values (971 ± 81 ng/ml). 5-FU concentrations were dose-dependent with low intra- and interindividual variability. However, care has to be taken, as the results can be influenced by inaccurate blood sampling: too early or late sampling (when the folfusor-pump is empty), delayed centrifugation of the tube or hemolysis. Conclusions: With critical analysis of the measurements and correct performance of blood sampling, the measurement of 5-FU plasma concentrations with the immunoassay may in the future allow to optimize 5-FU dosing and to identify the cause of toxicity. Changes of 5-FU clearance in long-term therapy still have to be studied.