Monkey B virus (Macacine herpesvirus 1; BV) is an alpha-herpesvirus of macaques that causes serious infections in humans. A spontaneous mutant resistant to penciclovir (PCV) was isolated. Several genes were sequenced to identify mutations potentially responsible for PCV resistance. A single nucleotide deletion in the thymidine kinase (TK) gene was identified. To confirm its role in PCV resistance, several TK recombinants were constructed. A TK-deletion virus and a recombinant carrying the mutation were both resistant to PCV, while a revertant was PCV-sensitive. These results demonstrate that spontaneous drug-resistant mutants of BV do occur and that the BV TK is responsible for sensitivity to PCV.