Neutrophils are immune cells that bind to, engulf, and destroy bacterial and fungal pathogens in infected tissue, and their clearance by apoptosis is essential for the resolution of inflammation. Killing involves both oxidative and nonoxidative processes, the oxidative pathway requiring electrogenic production of superoxide by the membrane-bound NADPH oxidase complex. A variety of stimuli, from bacterial chemotactic peptides to complement-or IgG-opsonized microbes, can induce the production of reactive oxygen species (ROS) by neutrophils, presumably by means of NADPH oxidase. We report here that 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of Ca 2؉ -activated potassium channels of small conductance (SK) and intermediate conductance (IK), causes production of superoxide and hydrogen peroxide by neutrophils and granulocyte-differentiated PLB-985 cells. This response can be partially inhibited by the SK blocker apamin, which inhibits a Ca 2؉ -activated K ؉ current in these cells. Analysis of RNA transcripts indicates that channels encoded by the SK3 gene carry this current. The effects of 1-EBIO and apamin are independent of the NADPH oxidase pathway, as demonstrated by using a PLB-985 cell line lacking the gp91phox subunit. Rather, 1-EBIO and apamin modulate mitochondrial ROS production. Consistent with the enhanced ROS production and K ؉ efflux mediated by 1-EBIO, we found that this SK opener increased apoptosis of PLB-985 cells. Together, these findings suggest a previously uncharacterized mechanism for the regulation of neutrophil ROS production and programmed cell death.N eutrophils, as key players in the innate immune response, must be able to identify, phagocytose, and neutralize a broad array of pathogenic microbes (1). Killing usually involves isolation of bacteria or fungi within the phagosome, although neutrophils can release toxic contents into their surroundings to kill extracellular microbes (1). These toxic contents include reactive oxygen species (ROS), such as hydrogen peroxide and hypochlorous acid, as well as proteases and antimicrobial peptides. Because these toxins also can damage host cells, limiting their release and clearing apoptotic neutrophils ultimately are necessary for resolving an infection (2). Understanding how neutrophils regulate the production of oxidants is, therefore, important for both resisting pathogens and controlling inflammation.Although ion channels are best known for their roles in neuronal and cardiac action potentials, their importance in some immune cells, particularly neutrophils, has begun to emerge over the past few years (1, 3-5). The NADPH oxidase of neutrophils produces large amounts of superoxide at the plasma and phagosomal membranes, resulting in an electrogenic efflux of electrons from the cytoplasm (6). An extensive literature indicates that a proton channel, whose molecular identity has recently been identified (7,8), carries a compensating outward current that prevents depolarization of the cell membrane to potentials that inhibit the NADPH oxidase...