Rationale: Reduced action potential duration (APD) and lack of APD rate dependent-adaptation are hallmarks of AF-induced electrical remodelling of the atrial myocardium. Understanding the mechanism underlying atrial electrical remodelling in AF is of fundamental importance for the prevention and treatment of AF. We have recently found that nitric oxide synthase (NOS) activity is dramatically reduced in atrial myocytes from goats and patients with AF. Whether loss of NOS activity contributes to the AF-induced atrial electrical remodelling remains to be established. Methodology: The whole cell current patch clamp technique was used to measure the AP in human and murine isolated atrial myocytes (N: number of patients or mice; n: number of myocytes). AF was induced in mice in vivo by using trans-oesophageal electrical stimulation. The protein content of constitutive NOSs (nNOS and eNOS) was assessed by Western blotting in atrial lysates. NOS activity was measured by the L-arginine to citrulline assay. Simulations were performed to evaluate the role of decreased nNOS activity in rotor stability in mouse right/left atrial (RA/LA) tissue models. Results: We found that decreased atrial NOS activity in patients with AF was associated with a significant reduction in nNOS protein expression (by 62%) whereas eNOS was unchanged. To evaluate the role of nNOS on atrial electrical properties, we examined the effects of the nNOS inhibitor, S-methylthiocitrulline (SMTC), or nNOS gene deletion on APD. SMTC induced 38%, 39% and 30% reduction in APD20, APD50 and APD90 respectively in RA myocytes from patients in Sinus Rhythm (SR) (N=8, n=38 control vs. N=8, n=31 cells perfused and dialyzed with SMTC, p<0.001) and suppressed the APD rate dependent-adaptation. In murine RA myocytes, inhibition and gene deletion of nNOS reduced APD by 38% and 22%, respectively (N=9, n=35 from nNOS-/- & N=6, n=11 wild type, WT, cells plus SMTC vs. N=10, n=28 control WT cells, p<0.001). SMTC had no effect on APD in atrial myocytes from nNOS-/- mice. nNOS inhibition and gene deletion also abolished the physiological RA to LA gradient in APD. In silico simulation indicated that lack of atrial APD gradient promotes rotor stability at the RA/LA junction which may contribute to increased AF propensity. In agreement with these findings, nNOS-deficient mice displayed a 2-fold increase in AF inducibility in response to stimulation (p<0.05 vs. WT littermates, N=18 in each group). Conclusions: In mammalian atrial myocytes, nNOS plays an important role in determining APD, its rate-adaptation and the physiological RA/LA electrical gradient. These findings suggest that the marked loss of nNOS protein and activity in the fibrillating atrial myocardium has potentially important implication for AF-induced electrical remodelling.