After translocation through the pleuristratified epithelium of the lower female genital tract, HIV-1 encounters potential target mononuclear cells in the lamina propria of the vagina and ectocervix. Here we show that each major type of genital mononuclear cells, including dendritic cells (DCs), macrophages and lymphocytes, are susceptible to HIV-1 in vitro. Among suspensions of vaginal and ectocervical mononuclear cells, DCs were the first cells to take up virus, containing GFP-tagged virions as early as 15 min after exposure. At 2 hr after exposure, DCs still contained the largest proportion of HIV-1(+) cells compared to lamina propria macrophages and lymphocytes from the same mucosal compartment. By 4 days, however, lymphocytes from both vaginal and ectocervical mucosa supported the highest level of HIV-1 replication. Genital macrophages from the same mucosal tissues also were permissive to HIV-1, in sharp contrast to intestinal macrophages, which we have shown previously do not support HIV-1 replication. Thus, among human vaginal and ectocervical mononuclear target cells, DCs are the first to take up HIV-1 and T cells support the most robust viral replication. Further characterization of the parameters of HIV-1 infection in genital mononuclear cells will enhance our understanding of HIV-1 infection in the female genital tract.