In order to observe the biodistribution of 5-fluorouracil (5-FU) and its main metabolites in different kinds of tissue (tumor, liver, kidney, spleen, mucosa, lungs, heart, peritoneum, pancreas) and serum according to various novel application forms, a simple and rapid method for the simultaneous determination of 5-FU and its active metabolites 5-fluorouridine (5-FUrd) and 5-fluoro-2'-deoxyuridine (5-FdUrd) has been established. Proteins in serum and tissue samples were precipitated by perchloric acid after addition of the internal standard 5-bromouracil. The compounds were separated using an ODS Hypersil (5 microm) column and detected by UV absorbance (254 nm). Specificity, linearity, reproducibility, intermediate precision and accuracy of the method were established. The lower limit of quantitation (LOQ) for the compounds in serum and various tissue samples was determined. Data on the recovery of the compounds and the internal standard are provided.