The dependence of human monocyte maturation in vitro on autologous serum was examined. If autologous serum was present during the monocyte culture, cytolysis of K562 target cells increased, intracellular levels of three lysosomal enzymes increased, and the fluoride-inhibitable esterase staining of the monocytes changed into a fluoride-resistant esterase stain (characteristic of more mature extravascular mononuclear phagocytes). Monocytes cultured in the presence and in the absence of serum also assumed different shapes. All of these changes were dependent on the concentration of autologous serum present (0-10%) and the length of time the monocytes were in culture (0-7 days). Lack of development by monocytes cultured in the absence of serum was not due to a general loss of the ability of these cells to function, because phagocytosis of antibody-coated erythrocytes was not lost following 7 days in culture in the absence of serum.