The cellular events that occur following occupational percutaneous exposure to HIV have not been defined. In this study, we studied relevant host cellular and molecular targets used for acquisition of HIV infection using split-thickness human skin explants. Blockade of CD4 or CCR5 before R5 HIV application to the epithelial surface of skin explants completely blocked subsequent HIV transmission from skin emigrants to allogeneic T cells, whereas preincubation with C-type lectin receptor inhibitors did not. Immunomagnetic bead depletion studies demonstrated that epithelial Langerhans cells (LC) accounted for > 95% of HIV dissemination. When skin explants were exposed to HIV variants engineered to express GFP during productive infection, GFP(+) T cells were found adjacent to GFP(+) LC. In three distinct dendritic cell (DC) subsets identified among skin emigrants (CD1a(+)langerin(+)DC-specific intercellular adhesion molecule grabbing non-integrin (SIGN)(-) LC, CD1a(+)langerin(-)DC-SIGN(-) dermal DC, and CD1a(-)langerin(-)DC-SIGN(+) dermal macrophages), HIV infection was detected only in LC. These results suggest that productive HIV infection of LC plays a critical role in virus dissemination from epithelium to cells located within subepithelial tissue. Thus, initiation of antiretroviral drugs soon after percutaneous HIV exposure may not prevent infection of LC, which is likely to occur rapidly, but may prevent or limit subsequent LC-mediated infection of T cells.