Placental macrophages (Hofbauer cells) were isolated and cultured in vitro to investigate their susceptibility to human immunodeficiency virus type 1(HIV-1) infection. Ofadherent cells, 80% expressed CDI4, and >99% were nonspecific esterase-positive. CD4 antigen was expressed at very low levels. CD4 mRNA could be detected in the cells by reverse transcription followed by polymerase chain reaction. The macrophages were infected productively after inoculation with low-passage blood isolates of cell-free HIV-1. Peak virus titers were detected 3–7 days after infection by HIV-l antigen ELISA and reverse transcriptase assay. Replication of HIV-l in placental macrophages was less than in blood monocytes. HIV-1 RNA was detected in placental macrophages by in situ hybridization 16 days after infection. Multinucleated giant cells were identified in some cultures, indicative of an HIV-induced cytopathic effect. Thus, placental macrophages can be infected productively with clinical isolates of HIV-1, and such cells may act as a reservoir of virus for transmission to the fetus in utero.