Reported herein are the results of studies demonstrating the utility of a chemically defined, serum-free medium designated as AIM-V (GIBCO) for the long-term (greater than 2 weeks) cultivation of functionally-defined human macrophages. The AIM-V medium is a mixture of HEPES-buffered Dulbecco's Modified Eagle Medium and Ham's Nutrient Mixture F12 that had been supplemented with purified human albumin, transferrin, insulin, and a proprietary mixture of purified factors. Nonadherent macrophages for serial studies were generated in petri dishes with a Teflon liner that had been seeded with a heterogeneous population of peripheral blood mononuclear cells of healthy human adults. For comparison, cultures were initiated with serum-free medium AIM-V and medium supplemented with AB/Rh+ serum or freshly collected autologous serum. Viability was defined by trypan blue dye, glass adherence, and phagocytosis of yeast. Macrophages were characterized by light microscopy, cytochemistry, and phenotypic analysis. Ultrastructural morphology was defined by scanning electron microscopy. These studies demonstrate that functionally defined human macrophages can be sustained in long-term culture with the use of serum-free medium that has not been augmented with mitogenic stimulants, growth-promoting lymphokines/monokines, or differentiation-inducing agents. Serum-free medium AIM-V, which has been approved for generating lymphokine, (i.e., interleukin-2; IL-2)-activated killer cells (LAK) for IL-2/LAK adoptive immunotherapy modalities, may also prove useful for studies defining and isolating regulatory proteins produced by activated monocytes and macrophages and for generating cytolytic macrophages for different antitumor regimens.