Syphilitic genital ulcers are cofactors for the bidirectional transmission of human immunodeficiency virus (HIV). U937 human promonocytic cells chronically infected with HIV-1 (Ul cells) or transiently transfected with wild type or mutant HIV long terminal repeat (LTR) reporter constructs were used to examine mechanisms that likely underlie Treponema pallidum-induced immune cell activation and consequent induction of HIV. Virulent T. pallidum, a representative native treponemal lipoprotein (NTp47), or synthetic lipoprotein analogues (lipopeptides) all induced HIV replication in U1 cells. These stimuli also induced HIV gene expression from a wild type HIV LTR. HIV gene expression correlated with the translocation of NF-κB, and mutations within the NF-κB binding sites of the HIV LTR abrogated HIV gene expression. This study implicates treponemal lipoproteins as key mediators of immune cell activation and provides insights into the cellular and molecular bases for enhanced HIV transmission in syphilitic persons.