Noninvasive methods for measuring the pharmacokinetics of chemotherapeutic drugs such as 5-fluorouracil (FU) are needed for individualized optimization of treatment regimens. PET imaging of 18FFU (PET/18FFU) is potentially useful in this context, but PET/18FFU is severely hampered by low tumor uptake of radiolabel and rapid catabolism of FU in vivo. Pretreatment with eniluracil (5-ethynyluracil) prevents catabolism of FU. Hypothesizing that suppression of catabolism would enhance PET/18FFU, we examined the effects of eniluracil on the short-term pharmacokinetics of the radiotracer. Anesthetized rats bearing a subcutaneous rat colorectal tumor were given eniluracil or placebo and injected intravenously 1 h later with 18FFU or 3HFU. In the 18F studies, dynamic PET image sequences were obtained 0-2 h after injection. Tumors were excised and frozen at 2 h and then analyzed for labeled metabolites by high-performance liquid chromatography. Biodistribution of radiolabel was determined by direct tissue assay. Eniluracil improved tumor visualization in PET images. With eniluracil, tumor standardized uptake values (activity/g/injected activity/g body weight) increased from 0.72 +/- 0.06 (mean +/- SEM; n = 6) to 1.57 +/- 0.20 (n = 12; P < 0.01), and tumor uptake increased by factors of 2 or more relative to plasma (P < 0.05) and bone, liver, and kidney (P < 0.01). Without eniluracil (n = 5), 57% +/- 4% of recovered radiolabel in tumor at 2 h was on catabolites, with the rest divided among FU (2% +/- 1%), anabolites of FU (38% +/- 7%), and unidentified peaks (4% +/- 2%). With eniluracil (n = 8), catabolites, FU, and anabolites comprised 2% +/- 1%, 41% +/- 5%, and 57% +/- 4%, respectively, of the recovered radiolabel in tumors. Eniluracil increased tumor accumulation of 18F relative to host tissues and fundamentally changed the biochemical significance of that accumulation. With catabolism suppressed, tumor radioactivity reflected the therapeutically relevant aspect of FU pharmacokinetics--namely, uptake and anabolic activation of the drug. With this approach, it may be feasible to measure the transport and anabolism of 18FFU in tumors by kinetic modeling and PET. Such information may be useful in predicting and increasing tumor response to FU.