AbstractParameters for inhibition of thymidylate synthase (TS) in a DMH-induced transplantable rat colon carcinoma were studied after intraperitoneal administration of bolus doxifluridine (5′-dFUR) 200mg/kg. Levels of 5′-dFUR, 5-fluorouracil (5-FU), fluorouridinediphosphate (FUDP), and fluorouridinetriphosphate (FUTP) were determined by use of high-performance liquid chromatography. Micromethods for analysis of 5-fluoro-2′-deoxyuridylate (FdUMP) and TS were used to study the in vivo intracellular pharmacokinetics of TS inhibition. Peak values of 5′-dFUR and 5-FU were found at 30 min and showed exponential declines with values close to zero at 5 hr. Substantial levels of FUDP and FUTP were found throughout the 24 h observation time. Peak FdUMP levels were modest compared to those observed after equimolar administration of 5-FU, but FdUMP persisted in amounts well above available binding sites on TS for the 24 h observation time. Reduction of free TS enzyme to undetectable levels (<0.05 pmol/g) lasted for 4 h, and at 24 h, there was still almost 70% enzyme inhibition. The total amount of TS (TStot) defined as free [3H]FdUMP-titrable enzyme (TSf) plus TS bound to FdUMP in a ternary complex (TSb) increased as a result of 5′-dFUR bolus injection from 15 to 50 pmol/g during the 24 hr observation time. We conclude from these data that 5′-dFUR is converted to 5-FU and subsequently to FdUMP, and the results suggest that 5′-dFUR exerts its cytotoxic effects through inhibition of TS and incorporation into RNA.