Production of superoxide (O(2)(·-)) by NADPH oxidases contributes to the development of hypertension and atherosclerosis. Factors responsible for activation of NADPH oxidases are not well understood; interestingly, cardiovascular disease is associated with both altered NADPH oxidase activity and age-associated mitochondrial dysfunction. We hypothesized that mitochondrial dysfunction may contribute to activation of NADPH oxidase. The effect of mitochondrial inhibitors on phagocytic NADPH oxidase in human lymphoblasts and whole blood was measured at the basal state and upon PKC-dependent stimulation with PMA using extracellular 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium or mitochondria-targeted 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine spin probes and electron spin resonance (ESR). Intracellular cytosolic calcium [Ca(2+)](i) was measured spectrofluorometrically using fura-2 AM. Incubation of lymphoblasts with the mitochondrial inhibitors rotenone, antimycin A, CCCP, or ruthenium red (an inhibitor of mitochondrial Ca(2+) uniporter) did not significantly change basal activity of NADPH oxidase. In contrast, preincubation with the mitochondrial inhibitors prior to PMA stimulation of lymphoblasts resulted in two- to three-fold increase of NADPH oxidase activity compared with stimulation with PMA alone. Most notably, the intracellular Ca(2+)-chelating agent BAPTA-AM abolished the effect of mitochondrial inhibitors on NADPH oxidase activity. Cytosolic Ca(2+) measurements with fura-2 AM showed that the mitochondrial inhibitors increased [Ca(2+)](i), while BAPTA-AM abolished the increase in [Ca(2+)](i). Furthermore, depletion of cellular Ca(2+) with thapsigargin attenuated CCCP- and antimycin A-mediated activation of NADPH oxidase in the presence of PMA by 42% and 31%, correspondingly. Our data suggest that mitochondria regulate PKC-dependent activation of phagocytic NADPH oxidase. In summary, increased mitochondrial O(2)(·-) and impaired buffering of cytosolic Ca(2+) by dysfunctional mitochondria result in enhanced NADPH oxidase activity, which may contribute to the development of cardiovascular diseases.