The antiviral activity of several nucleoside analogues is often limited by their rapid degradation by pyrimidine nucleoside phosphorylases. In an attempt to avoid this degradation, several modified nucleosides have been synthesized. A series of 4'-thio-2'-deoxyuridines exhibits an anti-[herpes simplex virus (HSV)] activity significantly higher (20-600 times) than that shown by the corresponding 4'-oxy counterpart. We investigated the mode of action of these compounds and we found that: (i) several 4'-thio-2'-deoxyuridines are phosphorylated to the mono- and di-phosphates by HSV-1 thymidine kinase (TK) more efficiently than their corresponding 4'-oxy counterpart; (ii) both are inhibitors of cellular thymidylate synthase; (iii) 4'-thio-2'-deoxyuridines are resistant to phosphorolysis by human thymidine phosphorylase; (iv) both 4'-oxy- and 4'-thio-2'-deoxyuridines are phosphorylated to deoxyribonucleotide triphosphate in HSV-1-infected cells and are incorporated into viral DNA; (v) 4'-thio-2'-deoxyuridines are better inhibitors than their 4'-oxy counterparts of [(3)H]thymidine incorporation in HSV-1-infected cells; (vi) 4'-thio-2'-deoxyuridines are not recognized by HSV-1 and human uracil-DNA glycosylases. Our data suggest that 4'-thio-2'-deoxyuridines, resistant to pyrimidine phosphorylase, can be preferentially or selectively phosphorylated by viral TK in HSV-infected cells, where they are further converted into triphosphate by cellular nucleotide kinases. Once incorporated into viral DNA, they are better inhibitors of viral DNA synthesis than their corresponding 4'-oxy counterpart, either because they are not recognized, and thus not removed, by viral uracil-DNA glycosylase, or because they preferentially interfere with viral DNA polymerase.