Activation of human immunodeficiency virus type 1 (HIV-1) provirus by herpes simplex virus type 1 (HSV-1) infection is mediated by both HSV-1 gene products and cellular transactivators. Previously, several key factors such as NF-κB-specific proteins p55 and p85, HLP-1 protein that binds to the LBP-1 sequences of the HIV-1 long terminal repeat (LTR) and the viral transactivator ICPO were found to play a role in the transcriptional activation of HIV-1 LTR expression. In this report, we describe binding of herpesvirus-specific protein TDP150 to the TAR DNA region of the HIV-1 LTR. Our data suggest that TDP150 may be related to the herpesvirus transactivator protein ICP4;both proteins are 150-kD DNA-binding proteins produced in HSV-infected cells in the presence of an inhibitor of viral DNA replication. However, the appearance of TDP 150-binding activity is delayed by several hours compared to that of ICP4 and the DNA-binding specificity of TDP 150 differs from that of purified ICP4. These results suggest that TDP 150 is not identical to ICP4;whether it is its analogue remains to be determined. TAR DNA alone can confer responsiveness of heterologous promoter to HSV-1 infection, suggesting that this region can function as an enhancer in HSV-1-infected cells. Deletion of the TAR region from the HIV-1 LTR has not changed significantly the HSV-1-mediated stimulation.