Seventy-nine nucleobase analogs were evaluated as potential inhibitors of Toxoplasma gondii uridine phosphorylase (UrdPase), and the apparent Ki (appKi) values for these compounds were determined. Based on the inhibition data, a structure-activity relationship for the binding of nucleobase analogs to the enzyme was formulated, using uracil as a reference compound. Two compounds were identified as very potent inhibitors of T. gondii UrdPase, 5-benzyloxybenzylbarbituric acid and 5-benzyloxybenzyluracil, which had appKi values of 0.32 and 2.5 microM, respectively. A comparison of the results from the present study, with similar studies on mammalian UrdPase and thymidine phosphorylase (dThdPase) (Niedzwicki et al., Biochem Pharmacol 32: 399-415, 1993) revealed that there are both similarities and differences between the catalytic site of T. gondii UrdPase and the catalytic sites of the mammalian enzymes with respect to binding of uracil analogs. One compound, 6-benzyl-2-thiouracil, was identified as a potent, specific inhibitor (appKi = 14 microM) of T. gondii UrdPase, relative to mammalian UrdPase and dThdPase.