The synthesis of a series of new C2-methyl-N10-alkylquinazoline-based thymidylate synthase (TS) inhibitors containing difluroinated p-aminobenzoate rings is described. Derivatives of the N10-propargyl and N10-methylquinazoline antifolates were prepared with 2',3'-, 2',5'-, and 2',6'-difluoro substitution. The synthesis of the 2',5'-difluoro analogues involved oxidation of the difluoronitrotoluene to 2,5-difluoro-4-nitrobenzoic acid followed by glutamation, reduction, and alkylation (propargyl bromide or MeI) to the diethyl N-(4-(alkylamino)-2,5-difluorobenzoyl)-L-glutamates. For the synthesis of the 2',3'- and 2',6'-difluoro compounds a new route was devised starting from methyl 4-((tert-butoxycarbonyl)amino)-2,6-difluorobenzoate and its 2,3-substituted counterpart. Treatment with NaH and then an alkyl halide introduced the N10-substituent. The methyl ester was hydrolyzed and the resulting acid was condensed with diethyl L-glutamate. The secondary amine was liberated using CF3CO2H and coupled with 6-(bromo-methyl)-3,4-dihydro-2-methyl-4-oxoquinazoline to yield the antifolate diesters. Final deprotection with mild alkali completed the synthesis in each case. The target compounds were tested as inhibitors of partially purified L1210 TS and also examined for their inhibition of the growth of L1210 cells in culture. Compared to their nonfluorinated parent compounds all the difluoro analogues were poorer inhibitors of TS. The greatest loss of enzyme activity was seen in the N10-propargyl analogues which contained one of the fluorine atoms ortho to the amine substituent. This loss was less apparent in the N10-methyl derivatives. Despite this lower inhibition of TS the majority of new compounds have equivalent cytotoxicity to their nonfluorinated predecessors.