Understanding the initial events in the establishment of vaginal human immunodeficiency virus type-1 (HIV-1) entry and infection has been hampered by the lack of appropriate experimental models. Here, we show in an ex vivo human organ culture system that upon contact in situ, HIV-1 rapidly penetrated both intraepithelial vaginal Langerhans and CD4(+) T cells. HIV-1 entered CD4(+) T cells almost exclusively by CD4 and CCR5 receptor-mediated direct fusion, without requiring passage from Langerhans cells, and overt productive infection ensued. By contrast, HIV-1 entered CD1a(+) Langerhans cells primarily by endocytosis, by means of multiple receptors, and virions persisted intact within the cytoplasm for several days. Our findings shed light on the very earliest steps of mucosal HIV infection in vivo and may guide the design of effective strategies to block local transmission and prevent HIV-1 spread.