(E)-5-(2-Bromovinyl)-2'-deoxyuridine is a potent antiherpes compound with far better activity against herpes simplex virus type 1 than type 2. To understand the role of drug metabolism in this differential antiviral activity, we examined the metabolic fate of this drug in virus-infected and mock-infected Vero cells by high-pressure liquid chromatography. After 8 h of incubation in which cells were exposed to 10 micrograms of the drug per ml, 63 pmol/10(6) cells of the parent compound was detected in acid-soluble extracts of mock-infected cells. Herpes simplex virus-infected cells, however, incorporated or metabolized, or both, up to 11,310 pmol/10(6) cells. Type 1-infected cells metabolized the drug to the triphosphate where as many as 5,565 pmol/10(6) cells were detected. In contrast, three strains of type 2-infected cells metabolized the drug to the monophosphorylated nucleotide and no further. The amount of drug getting into the cells was virus strain and inoculum dependent. These studies indicate that poor substrate acceptance of (E)-5-(2-bromovinyl)-2'-deoxyuridine monophosphate by herpes simplex virus type 2-specified thymidylate kinase is an important factor in situ in infected cells, preventing anabolism of the parent compound to its active triphosphorylated form. This may account for its type specificity.