Proteins encoded by a variety of DNA viruses activate gene expression from the promoter within the long terminal repeat (LTR) of the human immunodeficiency virus type 1 (HIV-1). The mechanism by which immediate-early (IE) gene products of human cytomegalovirus (CMV) activate expression from the HIV-1 LTR was examined in transient expression assays in cultures of human cells by using plasmids containing the LTR linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and a plasmid expressing the CMV IE gene. Analysis of clustered site mutations within the HIV-1 LTR revealed that sequences from nucleotides -6 to +20 (relative to the start site of transcription) are critical for responsiveness to transactivation by CMV IE gene products. This region partially overlaps the trans-acting response element (+19 to +42) required for function of the HIV-1 transactivator. The CMV IE gene was shown to increase the steady-state levels of both prematurely terminated and full-length transcripts initiated within the LTR. These results support a model in which CMV IE gene products act through a specific regulatory element in the HIV-1 LTR to increase viral transcription.