Abstract 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 ICPO and ICP4 are thought to be important mediators of this process, which is known to involve the induction of the cellular activators NF- κB and Sp1. We demonstrate that ICPO and ICP4 transactivation of the LTR is largely dependent on the presence of NF- κB and Sp1 binding sites. However, in Jurkat CD4-positive lymphocytes, HSV-1 activates LTR constructs lacking all NF- κB 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 ICPO and ICP4. Thus HSV-1 IE genes transactivate the HIV-1 LTR both through the induction of NF- κB and Sp1 and through another as yet undefined cellular factor.