The antiherpes agent 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) is a much more potent inhibitor of herpes simplex viruses in vivo than acyclovir, yet both are equally active in vitro against these viruses. To explain this difference, studies were conducted to compare the intracellular metabolism and enzymatic phosphorylation of the two compounds. In herpes type 1 and type 2 infected cells, the levels of DHPG triphosphate were only about 2-fold greater than levels of acyclovir triphosphate at virus-inhibitory concentrations (less than or equal to microM). At concentrations greater than 2.5 microM in herpes type 1 but not in type 2 infected cells, acyclovir phosphorylation was inhibited relative to that of DHPG. When drug was removed after 6 hr from infected cells, acyclovir triphosphate rapidly degraded to acyclovir and was excreted into the culture medium. In contrast, DHPG triphosphate persisted at 60-70% of the original level for 18 hr after drug removal, and DHPG excretion from cells was very slow. This finding could be a key factor to the superior potency of DHPG in animals, despite the fact that blood levels of both compounds fall rapidly after dosing. In uninfected cells, low levels of DHPG and acyclovir triphosphates were produced at 100 microM concentrations. Phosphorylation of DHPG to mono-, di- and triphosphates by purified viral and cell enzymes was more rapid than that of acyclovir. However, acyclovir triphosphate was a much more potent inhibitor of herpes virus and cell DNA polymerases.