9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a selective and potent inhibitor of retrovirus and herpesvirus replication in vitro and in vivo. In cell culture studies, pretreatment of HeLa S3 cells with PMEA before infection enhanced its antiviral potency by almost 10-fold, compared with treatment of the cells only after viral infection. To elucidate the basis for this observation, the uptake, metabolism, and retention of PMEA metabolites were examined in uninfected and herpes simplex virus type 1-infected cells, by using [2,8-3H]PMEA. Uptake of the drug into both acid-soluble and acid-insoluble fractions was slow and did not begin to plateau until close to 24 hr. High performance liquid chromatographic analysis of acid-soluble extracts revealed at least four metabolites in addition to PMEA itself, designated as X, Y, DP, and TP. Metabolites X and Y, which were distinct from PMEA and its mono- and diphosphoryl derivatives, represented almost 90% of the radioactivity associated with the cells after 24 hr of incubation. Dephosphorylation of acid-soluble metabolites resulted in accumulation of radioactivity in the peaks associated with PMEA and X. Most of the radioactivity in the acid-insoluble fraction was associated with DNA. Enzymatic digestion of [3H] PMEA-labeled DNA from either infected or uninfected cells yielded both metabolite X and PMEA itself. The role of newly discovered PMEA metabolites in its antiviral activity and cytotoxicity is not clear.