In cells infected with herpes simplex virus type 1, intracellular dNTP pools increased markedly. Treatment of these cells with 3 microM acyclovir resulted in an additional expansion in pyrimidine deoxyribonucleoside triphosphate pools with dTTP increasing 32-fold and dCTP 8-fold. Both thymidine and deoxycytidine, however, compete with acyclovir for phosphorylation by the viral pyrimidine deoxyribonucleoside kinase and thus reduce the amount of drug that is anabolized to the active form. Theoretically, agents which inhibit thymidylate synthase or dihydrofolate reductase should reduce intracellular pools of thymidine, resulting in the potentiation of the antiviral effects of acyclovir. We explored this strategy by quantitating the synergy produced by combinations of acyclovir and other drugs using three-dimensional dose-response surface methodology (MacSynergy II). Significant synergy was seen with both 5-FdUrd and methotrexate whereas BrVdUrd, 5-CldUrd, 5-IdUrd, and 5-BrdUrd exhibited little to no synergistic activity. It is suggested that inhibitors of thymidylate synthase and dihydrofolate reductase warrant further exploration as potentiators of acyclovir.