Sexual transmission of human immunodeficiency virus (HIV) accounts for the majority of new infections worldwide. However, the mechanism of viral transmission across the mucosal barrier is poorly understood. By use of an ectocervical epithelium-derived cell line, we found that the cells are capable of sequestering large amounts of HIV particles but are refractory to cell-free viral infection. The sequestered virus particles remained infectious for >/=6 days and resisted treatment with trypsin. Upon coculture with CD4(+)-susceptible cells, epithelial cells can effectively transmit the virus to these cells, which can result in robust infection of the target cells. Inhibitory studies have shown that heparan sulfate moiety of cell-surface proteoglycans is involved in the viral attachment to these CD4-negative epithelial cells. Genital epithelial cells may play active roles in sequestering, protecting, and transferring virus during sexual transmission of HIV.