Endothelial release of tissue-type plasminogen activator (t-PA) regulates fibrinolysis and is considered to be a primary endogenous defense mechanism against thrombosis. Adiposity is associated with an increased risk of atherothrombotic events. We determined the influence of overweight and obesity on the capacity of the vascular endothelium to release t-PA and the effects of regular aerobic exercise on endothelial t-PA release in previously sedentary overweight and obese adults. First, we studied 66 sedentary adults: 28 normal-weight (BMI <25 kg/m2); 22 overweight (BMI ≥25 and <30 kg/m2); and 16 obese (BMI ≥30 kg/m2). Net endothelial t-PA release was determined in vivo in response to intrabrachial infusions of bradykinin (BK) and sodium nitroprusside. Second, we studied 17 overweight and obese adults who completed a 3-mo aerobic exercise intervention. Net release of t-PA in response to BK was ∼45% lower ( P < 0.01) in overweight (from 0.1 ± 0.4 to 41.7 ± 4.9 ng·100 ml tissue−1·min−1) and obese (−0.1 ± 0.6 to 47.7 ± 5.2 ng·100 ml tissue−1·min−1) compared with normal-weight (0.1 ± 0.8 to 77.5 ± 6.7 ng·100 ml tissue−1·min−1) adults. There was no difference in t-PA release between the overweight and obese groups. Exercise training significantly increased t-PA release capacity in overweight and obese adults (from −0.3 ± 0.5 to 37.1 ± 4.9 ng·100 ml tissue−1·min−1before training vs. 1.0 ± 0.9 to 65.4 ± 6.3 ng·100 ml tissue−1·min−1after training) to levels comparable with those of their normal-weight peers. These results indicate that overweight and obesity are associated with profound endothelial fibrinolytic dysfunction. Importantly, however, regular aerobic exercise can increase the capacity of the endothelium to release t-PA in this at-risk population.