Varicella-zoster virus (VZV) causes chicken pox (varicella), becomes latent in dorsal root ganglia, and reactivates decades later to cause shingles (zoster). During latency, the entire VZV genome is present in a circular form, from which genes 21, 29, 62, and 63 are transcribed. Immediate-early (IE) VZV genes 62 and 63 encode regulators of virus gene transcription, and VZV gene 29 encodes a major DNA-binding protein. However, little is known about the function of VZV gene 21 or the control of its transcription. Using primer extensions, we mapped the start of VZV gene 21 transcription in VZV-infected cells to a single site located at -79 nucleotides (nt) with respect to the initiation codon. To identify the VZV gene 21 promoter, the 284-bp region of VZV DNA separating open reading frames (ORFs) 20 and 21 was cloned upstream from the chloramphenicol acetyltransferase gene. In transient-transfection assays, the VZV gene 21 promoter was transactivated in VZV-infected, but not uninfected, cells. Further, the protein encoded by ORF 62 (IE62), but not those encoded by VZV ORFs 4, 10, 61, and 63, transactivates the VZV gene 21 promoter. By use of transient-cotransfection assays in conjunction with 5' deletions of the VZV gene 21 promoter, a 40-bp segment was shown to be responsible for the transactivation of the VZV gene 21 promoter by IE62. This region was located at -96 to -56 nt with respect to the 5' start of gene 21 transcription.