5-Fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) is the active metabolite of the anticancer agent 5-fluorouracil (5FU). Antibodies against a conjugate of thyroglobulin with acetylated FdUMP were raised in 4 rabbits. The maximal titer was reached after 3-5 boosters. Subsequently, the antisera were collected and partially purified. In a competition assay a 100-fold excess of the natural nucleotide dUMP could displace tritiated FdUMP (20 pmol per assay) by about 50% in all antisera. However, tritiated dUMP itself did not bind to the antibody. No cross-reactivity was observed with the FdUMP precursor 5FU and with the natural nucleoside uridine and the nucleotides dTMP, dTTP and UTP. A considerable cross-reactivity was observed with the monophosphate of bromodeoxyuridine (Br-dUMP). Radio-immuno assays for FdUMP and dUMP were developed, for which a 100-fold dilution could be used. The FdUMP assay was linear in a range of 0.1 to 5 pmol FdUMP in aqueous solutions. Tumour samples contained a non-identified interfering factor; a similar interference was observed in an enzyme based assay for FdUMP. The dUMP assay was performed by competition of unlabeled dUMP with tritiated FdUMP and was linear from 50 to 2000 pmol dUMP per assay. The antibody recognized FdUMP bound in a ternary complex synthesized in a cell-free system between FdUMP, its target enzyme thymidylate synthase and the folate co-factor. Immunohistochemical staining for demonstration of the ternary complex in 5FU treated cells and tumours from patients and animals was not yet successful, neither with peroxidase nor with immunofluorescence staining. Possibly the amount of bound FdUMP is below the detection limit or FdUMP bound to TS is masked. The antibody may prove to be useful in studies on modulation of FdUMP and dUMP after treatment with thymidylate synthase inhibitors.