(E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU) is a highly selective inhibitor of herpes simplex virus-1 (HSV-1). A cell line (LH-1) expressing the HSV thymidine kinase (TK) gene is extremely sensitive to BVDU, whereas the parent line that expresses the cellular TK is resistant to BVDU. A TK-deficient derivative cell line (LMTK(-] which expresses neither the cellular nor viral TK was also extremely sensitive to BVDU. This suggests that the thymidine analog, BVDU, can be phosphorylated in thymidine kinase-deficient cells by pathways other than the cellular or HSV-encoded thymidine kinase.