Background - Atrial fibrillation (AF) is associated with an increased risk of stroke due almost exclusively to emboli from left atrial appendage (LAA) thrombi. Recently, we reported that AF was associated with endocardial dysfunction, limited to the left atrium (LA) and LAA and manifest as reduced nitric oxide (NO) production and increased expression of plasminogen activator inhibitor-1. We hypothesized that reduced LAA NO levels observed in AF may be associated with increased superoxide (O2.-) production. Methods and Results - After a week of AF induced by rapid atrial pacing in pigs, O2.- production from acutely isolated heart tissue was measured by 2 independent techniques, electron spin resonance and superoxide dismutase-inhibitable cytochrome C reduction assays. Compared with control animals with equivalent ventricular heart rates, basal O2 .- production was increased 2.7-fold (P<0.01) and 3.0-fold (P<0.02) in the LA and LAA, respectively. A similar 3.0-fold (P<0.01) increase in LAA O2.- production was observed using a cytochrome C reduction assay. The increases could not be explained by changes in atrial total superoxide dismutase activity. Addition of either apocyanin or oxypurinol reduced LAA O2.-, implying that NADPH and xanthine oxidases both contributed to increased O2.- production in AF. Enzyme assays of atrial tissue homogenates confirmed increases in LAA NAD(P)H oxidase (P=0.04) and xanthine oxidase (P=0.01) activities. Although there were no changes in expression of the NADPH oxidase subunits, the increase in superoxide production was accompanied by an increase in GTP-loaded Rac1, an activator of the NADPH oxidase. Conclusions - AF increased O 2.- production in both the LA and LAA. Increased NAD(P)H oxidase and xanthine oxidase activities contributed to the observed increase in LAA O2.- production. This increase in O2 .- and its reactive metabolites may contribute to the pathological consequences of AF such as thrombosis, inflammation, and tissue remodeling.