Herpes simplex virus 1 (HSV-1) infection induces transcription of the chloramphenicol acetyltransferase (CAT) gene directed by the long terminal repeat (LTR) of human immunodeficiency virus (HIV) in both transiently and permanently transfected cells containing the HIV-LTR/CAT hybrid gene. To define the mechanism by which HSV-1 stimulates the HIV LTR, we examined the effects of isolated regulatory genes from HSV-1. The results of cotransfection assays with the immediate-early (IE) genes of HSV-1, IE110 (ICP0) and IE175 (ICP4), showed that the IE110 protein, either alone or in combination with the IE175 protein, can activate the HIV LTR. Cotransfection with the IE175 gene alone or with the Vmw65 gene (coding for a virion transcription factor) alone did not lead to HIV-LTR activation. The lack of requirement for the IE175 or Vmw65 gene products in transient-expression assays was confirmed in permanent cell lines containing the HIV-LTR/CAT hybrid gene by using temperature-sensitive mutants defective in the IE175 gene product or in uncoating functions. By deletion analysis, we localized a 73-bp-long region (positions -104 to -32) from the HIV LTR that responded to HSV-1 activation; when this region, which is distinct from the previously identified trans-activating responsive (TAR) region, was ligated to a heterologous, HSV-1-nonresponsive gene (alpha 4-interferon/CAT), it conferred inducibility by both HSV-1 infection and IE110/175 cotransfection. Both simian and human cytomegalovirus also induced the HIV-LTR/CAT hybrid gene. However, we failed to detect specific upstream sequence requirements for induction by cytomegalovirus. Our results indicate that infection with unrelated viruses can alter the expression of HIV in an infected cell.