5-Ethynyluracil (776C85), a potent, mechanism-based, irreversible inactivator (Porter et al., J Biol Chem 267:5236-5242, 1992) of purified dihydropyrimidine dehydrogenase (DPD, uracil reductase, EC 1.3.1.2), readily inactivated DPD in vivo. DPD was assayed in tissue extracts by measuring the release of 14CO2 from [2-14C]uracil with an improved method. Specific activities from 0.1 to > 1000 U/mg protein were reproducibly measured. After rats were orally dosed with 20 micrograms/kg 5-ethynyluracil, liver, intestinal mucosa, lung, and spleen DPD were inactivated by 83-94%. The dose required to inactivate rat liver, rat brain, and mouse liver DPD by 50% was 1.8, 11, and 8.9 micrograms/kg, respectively. Rat liver DPD was inactivated completely within 25 min after an oral dose of 500 micrograms/kg 5-ethynyluracil. New DPD was synthesized with a half-time of 63 hr. We also developed an assay based on stoichiometric inactivation of DPD by 5-ethynyluracil to measure 5-ethynyluracil in plasma samples. Samples containing 5-ethynyluracil were incubated with rat liver extract for 24 hr at 12 degrees and then assayed for DPD. DPD activity decreased linearly with the concentration of 5-ethynyluracil (between 0 and 20 nM 5-ethynyluracil). The assay could detect 5-ethynyluracil at concentrations as low as 6 nM in human plasma and was not affected by high concentrations of uracil.