Human herpesvirus 6 (HHV-6) is a recently described T-cell pathogen whose medical relevance and molecular biology are just beginning to be addressed. As a first look at the regulation of viral genes, control of the HHV-6 DNA polymerase promoter was examined. Polymerase gene transcription in HHV-6-infected cells was found to initiate from a single site located 115 bases upstream of the translation start codon. A polymerase promoter-chloramphenicol acetyltransferase reporter gene construct failed to be expressed in uninfected T cells but was highly active in HHV-6-infected cells. Mutational data indicated that the polymerase promoter is TATA-less. Mutational analysis also revealed that the major upstream promoter regulatory element required for transcriptional activity in HHV-6-infected cells is a palindromic ATF/CREB transcription factor binding site. The significance of this site for promoter induction was further demonstrated by the fact that the polymerase ATF/CREB element, when appended to a heterologous basal promoter, is highly responsive to HHV-6 infection. Two protein complexes were found to bind in a specific manner to the ATF/CREB motif in both uninfected and HHV-6-infected T-cell nuclear extracts. Site-specific mutation of the ATF/CREB site resulted in loss of protein binding as well as loss of promoter activity in HHV-6-infected cells.