Short exposure of uninfected lymphocytes to UC781, a tight-binding nonnucleoside reverse transcriptase inhibitor (NNRTI), renders these cells refractory to subsequent HIV infection in the absence of exogenous drug (Borkow et al: J Virol 1997;71:3023-3030). Since epithelial cells may play a role in the sexual transmission of HIV-1, we examined the ability of NNRTI pretreatment to protect ME180 cervical epithelial cells and I407 intestinal epithelial cells from subsequent HIV-1 infection. Epithelial cells were pretreated with NNRTI, then exposed to HIV-1 chronically infected H9(+) cells for a short time following removal of the exogenous drug. The epithelial cells were productively infected by HIV-1, as shown by the presence of integrated HIV-1 proviral DNA, the presence of intracellular p24 antigen, and the production of nascent HIV-1 virions (cell-free p24) at various times postinfection. UC781 pretreatment of the epithelial cells did not prevent HIV-1 infection, since the cells had integrated proviral DNA, but the infectivity of virus subsequently produced from the UC781-treated cells was attenuated in a dose-dependent manner and virtually abolished at UC781 concentrations readily attainable in vivo. In contrast, nevirapine was ineffective in this respect, suggesting that not all NNRTI have microbicidal potential. The abrogation of infectivity of virus produced from UC781-pretreated epithelial cells suggests that this NNRTI may be useful in vaginal microbicide formulations targeted to inhibit HIV-1 in the vaginal/cervical or rectal milieus of a newly exposed individual.