Monostratified epithelial cells translocate HIV type 1 (HIV-1) from the apical to the basolateral surface via vesicular transcytosis. Because acutely transmitted HIV-1 is almost exclusively CCR5-tropic and human intestinal epithelial cells preferentially transcytose CCR5-tropic virus, we established epithelial monolayers using polarized HT-29 cells transduced to express CCR5, and an explant system using normal human rectal mucosa, to characterize biological parameters of epithelial cell transcytosis of HIV-1 and assess antiviral Ab blockade of transcytosis. The amount of cell-free HIV-1 transcytosed through the epithelial monolayer increased linearly in relation to the amount of virus applied to the apical surface, indicating transcytosis efficiency was constant (r2 = 0.9846; p < 0.0001). The efficiency of HIV-1 transcytosis ranged between 0.05 and 1.21%, depending on the virus strain, producer cell type and gp120 V1-V3 loop signature. Inoculation of HIV-1 neutralizing Abs to the immunodominant region (7B2) or the conserved membrane proximal external region (2F5) of gp41 or to cardiolipin (IS4) onto the apical surface of epithelial monolayers prior to inoculation of virus significantly reduced HIV-1 transcytosis. 2F5 was the most potent of these IgG1 Abs. Dimeric IgA and monomeric IgA, but not polymeric IgM, 2F5 Abs also blocked HIV-1 transcytosis across the epithelium and, importantly, across explanted normal human rectal mucosa, with monomeric IgA substantially more potent than dimeric IgA in effecting transcytosis blockade. These findings underscore the potential role of transcytosis blockade in the prevention of HIV-1 transmission across columnar epithelium such as that of the rectum. Copyright © 2010 by The American Association of Immunologists, Inc.