nef, the 3'-most open reading frame of HIV, has been reported to enhance HIV replication in various host cell types and to promote in vivo replication and pathogenesis. The mechanism underlying the increased in vivo viral replication is still unclear. We have examined the effect of a nef deletion on the infection of primary human CD4+ T lymphocytes and macrophages, using clones with nef and env sequences derived, respectively, from T cell- and macrophage-tropic viruses. The deletion of nef enhanced the formation of syncytia in CD4+ T lymphocytes infected with macrophage-tropic clones, despite a severalfold reduced viral production. No such enhancement of syncytium formation was observed in CD4+ T lymphocytes infected with a T cell line-tropic clone, but in this clone, the deletion of nef imparted a more severe replication defect. A similar increase in syncytium formation was observed in primary human macrophages infected with nef-deleted clones compared with wild-type counterparts, except under conditions in which the deletion of nef markedly reduced viral replication. We could not demonstrate an enhanced cell surface expression of HIV-1 envelope in lymphocytes infected with nef-deficient clones to explain the increased syncytium formation. In enhancing the HIV-1 cytopathic effect, the deletion of nef might curtail virus production by infected cells, and thus explain in part the reduced viral load observed in vivo in hosts infected with nef-deficient viruses.