To understand the mechanism of regulation of bovine herpesvirus 4 (BHV-4) early gene expression by immediate-early (IE) gene products, we studied activation of expression of the BHV-4 early gene encoding thymidine kinase (TK). BHV-4 encodes two IE RNAs. IE RNA 2 encodes a protein with predicted amino acid sequence similarity to the Epstein-Barr virus (EBV) R transactivator. The BHV-4 TK promoter-regulatory region was specifically transactivated approximately 9-fold by the IE2 gene product in transient expression cotransfection assays. Deletion analysis localized the IE2 responsive element(s) within a 72 bp fragment 5' to the start of transcription. Multiple copies of this 72 bp fragment conferred IE2-responsiveness on an enhancerless simian virus 40 (SV40) promoter and mediated IE2 transactivation in either orientation. Gel retardation assays demonstrated a sequence-specific complex between the IE2 gene product and the 72 bp DNA fragment, and indicated that the IE2 gene product binds DNA as a dimer. Sequences contained in a 25 bp subfragment were sufficient for IE2-DNA complex formation. No nucleotide sequence similarity was noted between the TK IE2-binding fragment and the consensus binding site for the EBV R transactivator. A single amino acid change in the putative DNA-binding domain of the IE2 gene product abolished both complex formation in vitro and transactivation activity in vivo.