Langerhans cells (LC) represent dendritic cells (DC) within mucosal epithelium that are purported initial targets for HIV following sexual exposure to virus. Here, morphologic, phenotypic, functional and HIV infection experiments were performed using monocyte‐derived DC cultured in the presence of GM‐CSF, IL‐4 and TGF‐β1 (G4T‐DC), GM‐CSF and IL‐4 (G4‐DC), and G4T‐DC incubated for an additional 3 days with CD40 ligand (CD40L‐DC). G4T‐DC, which demonstrated characteristics of immature LC, could be productively infected by either R5‐ or X4‐HIV strains. Infection levels, however, were markedly lower than those observed in immature G4‐DC. Surprisingly, CD40L‐DC, which demonstrated features of mature LC, could be productively infected with HIV at higher levels than immature G4T‐DC. Productive HIV infection in these three DC populations correlated positively with cell surface expression of CD4, CCR5 and CXCR4. We suggest that low levels of HIV infection in LC‐like G4T‐DC indicate an inefficient mechanism by which HIV can initially infect individuals, perhaps explaining the relative difficulty in becoming infected during sexual exposure to virus. In addition, enhanced HIV infection in LC‐like G4T‐DC following CD40L treatment suggests a mechanism by which inflammatory CD40L⁺ T cells, if present in mucosal tissue, could lead to increased HIV transmission rates.