Several soluble mediators, including endotoxin, prime neutrophils for an enhanced respiratory burst in response to subsequent stimulation. Priming of neutrophils occurs in vitro, and primed neutrophils are found in vivo. We previously localized the anion transporter ClC-3 to polymorphonuclear leukocytes (PMN) secretory vesicles and demonstrated that it is required for normal NADPH oxidase activation in response to both particulate and soluble stimuli. We now explore the contribution of the NADPH oxidase and ClC-3 to endotoxin-mediated priming. Lipooligosaccharide (LOS) from Neisseria meningitidis enhances the respiratory burst in response to formyl-Met-Leu-Phe, an effect that was impaired in PMNs lacking functional ClC-3 and under anaerobic conditions. Mobilization of receptors to the cell surface and phosphorylation of p38 MAPK by LOS were both impaired in PMN with the NADPH oxidase chemically inhibited or genetically absent and in cells lacking functional ClC-3. Furthermore, inhibition of the NADPH oxidase or ClC-3 in otherwise unstimulated cells elicited a phenotype similar to that seen after endotoxin priming, suggesting that basal oxidant production helps to maintain cellular quiescence. In summary, NADPH oxidase activation was required for LOS-mediated priming, but basal oxidants kept unstimulated cells from becoming primed. ClC-3 contributes to both of these processes.