Identification of the factors which impact on the transmission of human immunodeficiency virus type 1 (HIV-1) from an infected mother to her infant is essential for the development of effective strategies to prevent perinatal HIV-1 infection. The current study was designed to determine if unstimulated human neonatal cord blood mononuclear cells (CBMC) differ from adult peripheral blood mononuclear cells (PBMC) in susceptibility to HIV-1 infection. Both cell populations were challenged with two laboratory and two clinical HIV-1 isolates with different phenotypic properties. Infection was evaluated by quantitation of p24 antigen production and p24 antigen expression by an enzyme immunoassay and immunofluorescence, respectively. T-cell markers were determined by flow cytometry. Unstimulated CBMC were preferentially infected by macrophage-tropic, non-syncytium-inducing (non-SI) laboratory and clinical isolates, whereas PBMC were more susceptible to T-lymphotropic, SI HIV-1 strains. The macrophage-tropic strain HIV-1Ba-L replicated to 100-fold higher titers in CBMC than a similar inoculum of the SI isolate HIV-1LAI. The opposite occurred in unstimulated PBMC, which replicated HIVLAI to eightfold higher titers than the macrophage-tropic isolate. These findings indicate that a selection of viral phenotype may occur with unstimulated CBMC displaying a predominant susceptibility to infection by macrophage-tropic, non-SI HIV-1 strains and that this selection may influence mother-infant transmission of HIV-1.