Cells of the mononuclear phagocyte system arise from circulating blood monocytes. Upon emigration from the vasculature, monocytes differentiate into macrophages, a process that monocytes similarly undergo in vitro. We have established primary cultures from elutriated or adherence-purified blood monocytes and analyzed the antigenic modulation during monocyte to macrophage transformation, which could be followed by the expression of specific antigens and which required as yet unknown inducer signals present in the serum. It is shown that in the absence of serum monocytes only survive in vitro when cultured adherent to plastic but rapidly die in suspension culture. Starting at 0.5%, serum induced maturation dose-dependently, with the optimal concentration being 2 to 5%. Of those antigens not present on monocyte, the low-affinity Fc receptor (CD16), the α-chain of the vitronectin receptor (CD51), gp65-MAX.1, and gp68-MAX.3 were expressed only upon serum-induced macrophage differentiation, whereas the transferrin receptor (CD71), MAX.26, and to some degree also gp65-MAX.11 appeared to be independent of maturation and were also found on primary cultures of adherent monocytes under serum-free conditions. In addition, the rapid induction of HLA class II antigens (within 24 hr) was similar with and without serum, as was the continued high-density expression in long-term culture. The monocyte-specific CD14 antigen was down-regulated in the absence of serum but kept its level of expression on differentiated macrophages. In comparison, alveolar and peritoneal macrophages, respectively, differed in their antigenic phenotype: Alveolar macrophages expressed high HLA class II antigens but low CD14, whereas for peritoneal macrophages the opposite was found. Both interferon-γ and -α suppressed macrophage maturation in vitro but had contrary effects on HLA class II and CD16 expression: Interferon-γ up-regulated the two types of antigens, which, in contrast, were down-regulated by interferon-α.