A radioimmunoassay (RIA) for dUTP, with a sensitivity of 3.78 fmol, has been developed. The antibody cross-reacted with dTTP so that affinity purification of the immunoglobulin G fraction was required before its use in the RIA. Cross-reactivity with UTP and with mono- and diphosphodeoxyuridylates has necessitated respectively sodium periodate oxidation and anion exchange chromatography of cell extracts, prior to RIA quantitation of dUTP directly in fractions from the chromatography column. Mean recovery rate of a range of concentrations of extracted dUTP standard taken through the entire procedure is 63.7% (7.8-31.3 pmol dUTP) although at a lower concentration (3.11 pmol) the recovery was only 36.2%. Results are reproducible with CV values of between 3.1 and 9.5%. The assay has been used to assess the presence of dUTP in A549 human lung carcinoma cells exposed to the thymidylate synthase inhibitor CB3717. The high sensitivity of the quantitation step has made it possible to measure dUTP in relatively small numbers (10(6)) of cells.