A varicella-zoster virus (VZV) strain resistant to 5-iodo-2'-deoxyuridine (IdUrd) and 5-bromo-2'-deoxyuridine (BrdUrd) but sensitive to (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdUrd) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVdUrd) was isolated. The 2'-deoxythymidine (dThd) kinase of this mutant (Ito) strain was characterized; it was much less efficient in phosphorylating dThd, 2'-deoxycytidine, and BrdUrd than were the dThd kinases from wild-type (CaQu, Kobayashi) VZV strains. The Ito dThd kinase had a markedly decreased affinity for dThd, 2'-deoxycytidine, and BrdUrd but only a slightly decreased affinity for IVdUrd than had the wild-type VZV dThd kinase. BrdUrd was incorporated to a much lesser extent in VZV (Ito strain)-infected cells than wild-type VZV-infected cells, but IVdUrd was incorporated in Ito VZV-infected cells as efficiently as in wild-type VZV-infected cells. While resistant to IdUrd and BrdUrd, the Ito strain was susceptible to inhibitors of de novo thymidylate biosynthesis such as aminopterin.