Cryopreservation of nucleated blood and bone marrow mononuclear cells has been used both to preserve such cells for clinical, diagnostic, or research use and to eliminate them as passengers in frozen tissue destined for transplantation. Although techniques for macrophage and monocyte cryopreservation have been described, little work has been done on the functional state of these cells following frozen storage. Using rabbit alveolar macrophages (AM), we have shown that AM can be frozen, stored, and recovered without morphologic changes. Furthermore, freshly isolated and cryopreserved AM do not differ in their adherence characteristics or the organization of their actin cytoskeleton. Cryopreserved normal AM also retained inducible functions such as superoxide anion (O2) release and production of tumor necrosis factor (TNF) and interleukin-1 (IL-1). In order to further define the effects of cryopreservation on macrophage biology, we have also frozen macrophages which have been primed in vitro, and have demonstrated that the primed state remains at prefreezing levels after thawing and subsequent analysis. These data help define the effects of cryopreservation on cell function and establish parameters for the clinical and experimental evaluation of cryopreserved mononuclear phagocytes. © 1995 by Academic Press, Inc.