Human first-trimester placental macrophages were identified in frozen tissue sections and following cultivation as single-cell suspensions. In placental sections, the placental macrophages were shown to be the main cell type of the stromal zone. They clearly expressed macrophage-specific antigen, identified by EBM 11 monoclonal antibody; all were positive for nonspecific esterase (NSE), and 40-60% of them expressed the HLA-DR antigen. These cells were negative to anti-alpha-human chorionic gonadotropin (HCG) monoclonal antibody. After preparation of the placental single-cell suspension, the macrophages comprised about 15% of the total cell population. This fact was established by the following findings: 15% of the placental cell suspension expressed the antigen recognized by the OKM5 antibody, 16% expressed the EBM 11 antigen, 13% expressed the HLA-DR antigen, 5.5% were positive for NSE, and 24% phagocytosed Staphylococcus albus. Thirty percent of the cells were positive to anti-alpha-HCG antibody. After 48 h of cultivation in defined tissue culture conditions, the macrophages comprised more than 95% of the culture cell population. In the 48-h cultures, 98% of the cells expressed the macrophage antigens identified by OKM5 and EBM 11 monoclonal antibodies, and 75% expressed the HLA-DR antigen; 95% were positive for NSE, 88% of the cells had Fc receptors, and 95% of them were able to phagocytose S. albus. Only 0.1% of the culture cell population were positive to anti-alpha-HCG monoclonal antibody. These assays showed that the placental macrophages are the main cells in the placental stroma. In our tissue culture conditions, incubation time was shown to be the dominant factor in selecting macrophages over other cell types, most probably because the macrophages are the only placental cells that have the ability to adhere to plastic dishes. This observation might also explain the contradictory results in various studies over the last decade, regarding the incidence of the placental mononuclear macrophages.