Dendritic cells (DCs) are thought to play a crucial role in the pathogenesis of HIV-1 infection. DCs are believed to transport virus particles to lymph nodes before transfer to CD4(+) lymphocytes. We have investigated the role of Nef in these processes. HIV-1 replication was examined in human immature DC-lymphocyte cocultures and in DCs or lymphocytes separately. Using various R5-tropic and X4-tropic HIV-1 strains and their nef-deleted (Deltanef) counterparts, we show that Nef is required for optimal viral replication in immature DC-T cells clusters and in T lymphocytes. Nef exerts only a marginal role on viral replication in immature DCs alone as well as on virion capture by DCs, long-term intracellular accumulation and transmission of X4 strains to lymphocytes. We also show that wild-type and Deltanef virions are similarly processed for MHC-I restricted exogenous presentation by DCs. Taken together, these results help explain how HIV-1 Nef may affect viral spread and immune responses in the infected host.