Herpes simplex virus type 1 (HSV-1) activates transcription from the long terminal repeat (LTR) promoter region of the human immunodeficiency virus type 1 (HIV-1). HSV-1 immediate-early (IE) genes ICP0 and ICP4 are thought to be important mediators of this process, which is known to involve the induction of the cellular activators NF-kappa B and Sp1. We demonstrate that ICP0 and ICP4 transactivation of the LTR is largely dependent on the presence of NF-kappa B and Sp1 binding sites. However, in Jurkat CD4-positive lymphocytes, HSV-1 activates LTR constructs lacking all NF-kappa B or Sp1 Binding sequences. This effect is still evident when all sequences upstream of the TATA motif are removed. Such enhancer-independent transactivation can be produced by cotransfection of ICP0 and ICP4. Thus HSV-1 IE genes transactivate the HIV-1 LTR both through the induction of NF-kappa B and Sp1 and through another as yet undefined cellular factor.