The synthesis of two new pteridine-7-spirocyclopropanes that are time-dependent irreversible inhibitors of dihydrofolate reductase is described. Several related compounds including a cyclopropyl substituted quinoxaline and 2-aminopteridine-4(3H),6(5H)-dione were also prepared but these compounds were not found to be time-dependent inhibitors. The inhibitors were assessed using several dihydrofolate reductases, Escherichia coli RT/39, two mutants of the E. coli enzyme, Lactobacillus casei, and chicken liver. Activity was found against all enzymes tested but most strongly against E. coli wild type enzyme.