The baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) expresses several immediate early genes in infected cells. One of these, ie2 (formerly called IEN), was mapped and identified by its ability to augment IE1-mediated transactivation of the delayed early 39k gene. IE2 also stimulated the expression of reporter genes linked to the ie1 promoter. To test whether the increase in expression was due to increased mRNA, primer extension analyses were performed. Spodoptera frugiperda cells were transfected with a plasmid encoding IE1 in the presence or the absence of a plasmid encoding IE2, and total cellular RNA was purified. Quantitative primer extension assays revealed that cells cotransfected with pIE1 and pIE2 contained higher levels of IE1 mRNA than cells transfected with pIE1 alone. To demonstrate directly that IE2 is a transcriptional regulator, in vitro transcription assays were performed. Nuclear extracts were prepared from S. frugiperda cells transfected with either pIE2 or pUC18. The extracts containing IE2 were more active than the control extracts in transcription of a C-free cassette linked to the IE1 promoter. Immunodepletion of IE2 from the transfected extracts reduced the transcription activity to that of the control extracts. Nuclear extracts prepared from IE2-transfected cells were also more active in transcription from the adenovirus major late promoter. Taken together, these results demonstrate that IE2 is a transcriptional regulator.