This study describes the isolation and characterization of human fetal Kupffer cells. We demonstrated that these cells have the potential to respond to cytokines and lipopolysaccharide with an increased production of tumor necrosis factor-alpha and interleukin-1 beta. Kupffer cells were characterized by: (1) morphologic characteristics after adherence to plastic, (2) staining for alpha-naphthyl acetate esterase, (3) immunofluorescence with monoclonal antibodies, and (4) phagocytosis of latex beads. More than 90% of the adherent cells were identified as macrophages. Kupffer cells cultured with lipopolysaccharide were able to produce interleukin-1 beta and tumor necrosis factor-alpha in a time- and dose-dependent fashion and maximal secretion was observed with the use of 10 micrograms of lipopolysaccharide per milliliter within 8 hours of treatment. We have demonstrated mature functional activity of human fetal Kupffer cells at an early gestational age (13 to 19 weeks) and discussed the roles that these cells may play in development and protection of the fetus.