The assessment of tissue-specific pharmacodynamics is desirable in the development of tumor-targeted therapies. Plasma deoxyuridine (dUrd) levels, a measure of systemic thymidylate synthase (TS) inhibition, has limited application for studying the pharmacodynamics of novel TS inhibitors targeted to the high affinity alpha-folate receptor (FR). Here, we have evaluated the utility of [(18)F]fluorothymidine positron emission tomography ([(18)F]FLT-PET) for imaging the tissue pharmacodynamics of BGC 945, an FR-targeted antifolate TS inhibitor; the nontargeted antifolate BGC 9331 was used for comparison. TS inhibition by both drugs induced a concentration-dependent increase in [(3)H]thymidine uptake in FR-positive human epidermoid KB cells. Membrane-associated equilibrative nucleoside transporter type 1 levels increased from 55,720 +/- 6,101 to 118,700 +/- 5,193 and 130,800 +/- 10,800 per cell at 100 mug/mL of BGC 9331 and BGC 945, respectively, suggesting this as a potential mechanism of increased nucleoside uptake. In keeping with these in vitro findings, tumor [(18)F]FLT accumulation in KB xenografts increased by >/=2-fold after drug treatment with maximal levels at 1 to 4 hours and 4 to 24 hours after BGC 9331 and BGC 945 treatment, respectively. Of interest to FR targeting, BGC 9331, but not BGC 945, induced accumulation of [(18)F]FLT uptake in intestine, a proliferative and TS-responsive tissue. For both drugs, quantitative changes in tumor [(18)F]FLT uptake were associated with increased tumor dUrd levels. In conclusion, we have validated the utility of [(18)F]FLT-PET to image TS inhibition induced by antifolates and shown the tumor-specific activity of BGC 945. This imaging biomarker readout will be useful in the early clinical development of BGC 945.