The conditions required for the production of varicella-zoster virus (VSV)-induced deoxythymidine kinase (dTk) have been studied. Extracts from Vero cells harvested 62 h after VZV infection were found to contain VZV-induced dTk activity, with a minimal contribution from the cellular dTk activity. VZV dTK was shown to have a broad substrate specificity phosphorylating both deoxythymidine, deoxycytidine, and iododeoxyuridine. Deoxythymidine triphosphate inhibition studies revealed an intermediate deoxythymidine triphosphate sensitivity when compared with that of the cellular cytosolar enzyme and the deoxythymidine triphosphate-insensitive herpes simplex virus dTk. An assay for VZV dTk-blocking antibodies was developed, with [125I]iododeoxyuridine as a substrate in the presence of a deoxythymidine triphosphate concentration which selectively blocked the dTK of host cell origin. A total of 79 serum samples were studied; these included serum pairs from patients with varicella or herpes zoster and single sera from immune and nonimmune adults. VZV dTk blocking antibodies were detected exclusively in sera from patients with herpes zoster. All serum pairs showing VZV dTK seroconversion also showed a parallel conversion of complement fixation titers. The VZV dTk antibodies were found to be of the immunoglobulin G class. The immunological specificity of VZV dTK was investigated, and no cross-reactivity with herpes simplex virus type 1 or 2 dTk was found.