Granulocytes generate a "respiratory burst" of NADPH oxidase-dependent superoxide anion (O(2)(-*)) production that is required for efficient clearance of bacterial pathogens. Hv1 mediates a voltage-gated H(+) channel activity that is proposed to serve a charge-balancing role in granulocytic phagocytes such as neutrophils and eosinophils. Using mice in which the gene encoding Hv1 is replaced by beta-Geo reporter protein sequence, we show that Hv1 expression is required for measurable voltage-gated H(+) current in unstimulated phagocytes. O(2)(-*) production is substantially reduced in the absence of Hv1, suggesting that Hv1 contributes a majority of the charge compensation required for optimal NADPH oxidase activity. Despite significant reduction in superoxide production, Hv1(-/-) mice are able to clear several types of bacterial infections.