Large quantities of human blood-derived monocytes have been cultured in suspension in nonadherent cell culture bags and maintained for up to 3 weeks in a serum-free medium. This serum-free medium contained Iscove's modified Dulbecco's medium (IMDM) supplemented with human albumin, alpha-phosphatidylcholine, transferrin, and insulin. Morphology, cell surface antigens, and functional properties of these in vitro maturing macrophages were studied in comparison with macrophages cultured in a standard medium containing 10% fetal calf serum. In this report we demonstrate that this serum-free medium allows a better yield of cell survival than the standard medium; it also allows the differentiation of blood monocytes into fully functional macrophagic cells that express the different antigens found in mature macrophages. The results indicate that the use of serum-free defined medium offers good conditions in which to culture large numbers of human monocytes and allows an accurate analysis of the effect of supplementation with growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) on the differentiation and survival of monocytes and macrophages. Serum-free cultures could also be helpful for the precise analysis of the cell secretion activity and for determining the factors that are responsible for monocyte maturation into macrophages.