We report the discovery of a fourth Sp1 binding site at the 5' end of the U3 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (HIV-1 Sp1 IV), localized to HXB2 nucleotides -433 to -441. This site is shown to bind Sp1 protein specifically in electrophoretic mobility shift assays. Sp1 protein appears to bind to HIV-1 Spl IV with 5 to 10 times lower affinity than to a consensus Sp1 site. Mutation of HIV-1 Sp1 IV in an HXB2-derived long terminal repeat-chloramphenicol acetyltransferase reporter construct gave no significant change in positive-sense transcription but abolished both basal and phorbol myristate acetate-activated negative-sense transcription. Taken together, the results further define the HIV-1 negative-sense promoter as an Sp1-dependent, phorbol myristate acetate-responsive, and Tat-inhibited promoter initiating at HXB2 nucleotide -450.