Plasma total and low-density lipoprotein (LDL) cholesterol are established risk factors for atherosclerotic vascular disease and may also contribute to a prothrombotic risk via enhanced platelet reactivity. This study examines whether high-density lipoprotein (HDL) cholesterol, which is inversely correlated with coronary artery disease, is associated with a reduced thrombogenic potential. Platelet thrombus formation was evaluated by exposing porcine aortic media placed in Badimon perfusion chambers to flowing nonanticoagulated venous blood for 5 minutes at a shear rate of 1,000 s⁻¹. Forty-five subjects, 23 normal (LDL 104 ± 31, HDL 50 ± 15 mg/dl) and 22 hypercholesterolemic (LDL 181 ± 45, HDL 41 ± 10 mg/dl) patients without coronary artery disease were studied. Platelet aggregation and CD62 antigen expression, and assay for circulating prothrombotic factors were also performed. In univariate analysis platelet thrombus formation correlated with weight (r = 0.33, p = 0.03), diastolic blood pressure (r = 0.39, p = 0.01), HDL cholesterol (r = −0.45, p = 0.003), total/HDL cholesterol (r = 0.43, p = 0.004) and LDL/HDL (r = 0.38, p = 0.01) ratios, and platelet CD62 expression (r = 0.41, p = 0.02). In multiple regression analysis only HDL cholesterol showed significant correlation with platelet thrombus formation (p = 0.03). Platelet aggregation and circulating prothrombotic factors did not correlate with platelet thrombus formation. A comparison between normal and hypercholesterolemic subjects revealed enhanced thrombus area (0.026 ± 0.20 vs 0.045 ± 0.039 mm²/mm; p = 0.04), resting CD62 expression (6 ± 7% vs 15 ± 10% positive platelets, p = 0.02), and platelet aggregation (16.7 ± 5.2 vs 21.7 ± 6.7 ohms, p = 0.04) in hypercholesterolemic subjects. Our results demonstrate that HDL cholesterol is a significant independent predictor of ex vivo platelet thrombus formation.