The human placenta is relatively resistant to Human immunodeficiency virus 1 (HIV-1), but obstetric complications associated with inflammatory processes, including chorioamnionitis and spontaneous preterm delivery, are associated with increased rates of vertical transmission. It was hypothesized that the pro-inflammatory mediator tumour necrosis factor alpha (TNF-α), which promotes HIV-1 transmission across endothelial membranes, increases HIV-1 transmission across the placenta. Flow cytometry and immunostaining studies were performed, which demonstrated that the HIV-1 receptors CD4, CCR5 and CXCR4 were not expressed by villous trophoblast cells. Consequently, primary villous trophoblast cells were not infected with cell-free HIV-1 isolates, as measured by in situ PCR and quantitative PCR, but villous trophoblast cells were infected by HIV-1-infected peripheral blood mononuclear cells (PBMC). HIV-1 from infected PBMC was rapidly transported across confluent transformed trophoblast cell monolayers by transcytosis, and TNF-α significantly upregulated transcytosis of HIV-1 across the trophoblast layer without disrupting cell viability or confluency. Inhibitors of TNF-α (antibodies against TNF-α and TNF-α receptors) and an anti-inflammatory drug (tenidap) significantly reduced transcytosis rates. It was concluded that the villous trophoblast is resistant to infection by cell-free HIV-1 but susceptible to transcytosis of HIV-1 from infected PBMC, and inflammatory mediators such as TNF-α may play a critical role in promoting maternal-fetal transmission of HIV-1.