A set of free energy calculations were performed for selected antifolate compounds as inhibitors of thymidylate synthase (TS). These calculations reproduced the nonadditive behavior of different substitutions on selected compounds described in the experiments by Jones et al(1). (J. Med. Chem. 1996, 39 (4), 904-17). Molecular dynamics (MD) simulations showed that the nonadditive behavior was due to the interference between the way different substituents interacting with key protein side chains. Pictorial representation of free energy change (PROFEC) and free energy calculations suggested that a compound not previously considered would bind more tightly to TS than the best binding known compounds in this series of propargyl inhibitors and, thus, could be a promising candidate in anticancer drug design.