Atrial fibrillation (AF) is the most common arrhythmia associated with increased risk of stroke. Endocardial expression of nitric oxide synthase (eNOS) may be dependent on laminar flow and cyclical stretch of the atrium. We hypothesized that decreased bioavailability of nitric oxide (NO) is associated with increased thrombogenicity in atrial appendage of patients with AF. Studies were performed in human umbilical vein endothelial cells (HUVEC), porcine right atria, and human right atrial appendage obtained at the time of coronary artery bypass grafting. HUVEC were treated with the eNOS inhibitor N(G)-nitro-L-arginine-methyl-ester (L-NAME), rinsed, and incubated with fluorescently labeled human platelets. As compared to control HUVEC, L-NAME treatment increased adhesion of platelets to HUVEC by 13%. In contrast, pretreatment of HUVEC with the calcium ionophore A23187 to activate eNOS, decreased adhesion of platelets by 27%, as compared to control HUVEC. Similarly, adhesion of platelets to porcine right atrial endocardial surface was enhanced by pretreatment of the atria with L-NAME, and reduced by pretreatment of atria with A23187. Next we examined the effect of L-NAME on HUVEC expression of tissue factor (TF), an important initiator of intravascular thrombin and fibrin formation, and plasminogen activator inhibitor-1 (PAI-1). Compared to untreated cells, L-NAME increased HUVEC expression of TF (0.03 ± 0.01 vs. 0.12 ± 0.06, control vs. L-NAME) and PAI-1 (1.33 ± 0.80 vs. 2.55 ± 1.86, control vs. L-NAME), as measured by real time PCR normalized to 18S rRNA. We next measured expression of eNOS, TF, and PAI-1 by real time PCR in right atrial appendage obtained from patients with sinus rhythm (age 71 ± 10 yrs, n = 8) or AF (age 75 ± 5 yrs, n = 6). Expression of eNOS was similar (0.05 ± 0.02 vs.0.04 ± 0.01), whereas expression of TF (0.22 ± 0.05 vs. 0.51 ± 0.24) and PAI-1 (0.0002 ± 0.0001 vs. 0.0015 ± 0.0008) were increased, in right atrial appendage from patients with AF. In summary, (1) adhesion of platelets to HUVEC and atrial endocardial surface is dependent on NO bioavailability, (2) depressed nitric oxide levels increase HUVEC expression of TF and PAI-1, and (3) there is a trend towards increased expression of TF and PAI-1 in atria of patients with AF. Endocardial dysfunction and decreased NO may be associated with enhanced platelet adhesion and thrombogenesis in patients with AF.