Considerable evidence suggests that sexual transmission of human immunodeficiency virus (HIV) is mediated via mononuclear cells that can infect epithelia of the genital tract. We describe here an in vitro model that can be used to examine the mechanism of cell-to-cell transmission of this virus. We have employed the system to identify agents that may be effective in a vaginal formulation to prevent HIV transmission via sexual contact. We have previously shown that chronically HIV-infected mononucleocytes can infect CD4-negative epithelial monolayers in the following manner: adhesion, via multiple microvilli, of HIV-infected mononucleocyte-derived cells to epithelial monolayers activates rapid virion secretion. Virions are then shed from the attached surface of the infected lymphocyte into a partially enclosed, microvilli-laden space between the cells. The shedding results in uptake of the virus and epithelial cell infection as demonstrated by ultrastructural examination and in vitro virological techniques. In this report, we present evidence from time-lapse films that HIV-infected lymphocytes adhere to the epithelium for a few minutes and then shift position to another site on the epithelium. As a result, one infected lymphocyte appears to be able to sequentially infected several cells of the epithelial monolayer. Using a fluorescence-based cell-cell adhesion assay to examine the effect of seminal fluid and a variety of chemical compounds on lymphocyte-to-epithelial adherence, we found that seminal fluid significantly increases the number of lymphocytes adhering to epithelia. This suggests that semen can serve as an effective medium for cell-cell transmission of HIV. On the other hand, sulfated polysaccharides and glutathione effectively inhibit cell-cell adhesion. Since the cell-cell adhesion step is critical to epithelial cell infection by HIV, these results suggest that anti-cell adhesion compounds may be effective in a vaginal formulation to reduce the probability of HIV infection.