Lysis of human culture-derived macrophages by extracellular ATP has recently been described, and treatment of macrophages with interferon-gamma rendered those cells significantly more sensitive to lysis. In addition, cell death occurred more rapidly in interferon (IFN)-treated cells than in untreated macrophages. In an attempt to identify the mechanism by which extracellular ATP affects macrophages, as well as to explore the differences between interferon-gamma-treated and untreated macrophages, selected metabolic inhibitors were included in the lytic assays. Of the compounds tested, three antagonists of calmodulin-linked pathways (trifluoperazine, KN-62, and calmidazolium) blocked the ATP-mediated lysis of both interferon-gamma-treated and colony-stimulating factor-treated macrophages in a dose-dependent manner. Early signals of the ATP ligation of the P2Z purinoceptors of human macrophages included increases in cytosolic [Ca2+] and depolarization of the plasma membrane. However, the inclusion of calmodulin antagonists in these assays did not abrogate either effect. These results suggest that the mechanism which mediates the efflux of 51Cr-labeled proteins from ATP-lysed macrophages is distinct from calcium mobilization and membrane depolarization, and may involve the generation of secondary pores/channels in the plasma membrane via a calmodulin-linked pathway.