Antifolates are used in the treatment of various human malignancies and exert their cytotoxic activity by inhibiting folate-dependent enzymes resulting in disruption of DNA synthesis and cell death. Here we devised a computerized hybrid functional petri nets (HFPN) modelling of folate metabolism under physiological and antifolate inhibitory conditions. This HFPN modelling proved valid as a good agreement was found between the simulated steady-state concentrations of various reduced folates and those published for cell extracts; consistently, the simulation derived total folate pool size (11.3 microM) was identical to that published for cell extracts. In silico experiments were conducted to characterize the inhibitory profile of four distinct antifolates including methotrexate (MTX), tomudex, and LY309887, which inhibit dihydrofolate reductase (DHFR), thymidylate synthase (TS) and glycineamide ribonucleotide transformylase (GARTFase), respectively, as well as pemetrexed which has the capacity to inhibit all three enzymes. In order to assess the inhibitory activity of antifolates on purines and pyrimidines, the biosynthesis rates of IMP (20.53 microM/min) and dTMP (23.8 microM/min) were first simulated. Whereas the biochemical inhibitory profile of MTX was characterized by increased dihydrofolate and decreased tetrahydrofolate (THF) concentrations, the remaining antifolates did not decrease THF levels. Furthermore, MTX was 766- and 10-fold more potent in decreasing the production rates of IMP and dTMP, respectively, than pemetrexed. LY309887 indirectly decreased the rate of dTMP production by reducing the levels of 5-CH2-THF, a folate cofactor for TS. Surprisingly, pemetrexed failed to inhibit DHFR even at high concentrations. This HFPN-based simulation offers an inexpensive, user-friendly, rapid and reliable means of pre-clinical evaluation of the inhibitory profiles of antifolates.