Virus-enhancing factors present in the female genital tract may influence the transmission of human immunodeficiency virus type 1 (HIV-1). Previously, the presence of a heat-stable soluble factor in the cervicovaginal lavage (CVL) fluid of both HIV-infected and -uninfected women that induces HIV-1 expression in T cells and monocytes was reported. Now this CVL factor was shown to increase HIV-1 gene expression through the activation of the κB enhancer in the viral long terminal repeat (LTR). DNA binding studies, together with functional studies using mutant LTR reporter constructs, indicate the requirement for an NF-κB-dependent pathway in the CVL-mediated activation of HIV-1 expression. CVL samples that activated HIV-1 expression also stimulated AP-1-dependent transcription. These data demonstrate that an HIV-inducing factor, distinct from heat-labile cytokines, present in the female genital mucosa can activate AP-1 and NF-κB and increase HIV-1 gene expression through the κB enhancer, possibly contributing to HIV-1 transmission.