PCSK9 is a natural inhibitor of the LDL receptor. Gain-of-function mutations may cause the familial hypercholesterolemia phenotype, whereas loss-of-function variants associate with reduced LDL-C levels and lower coronary risk. Statins up-regulate PCSK9 in hepatocytes. We developed an assay to measure total PCSK9 in human plasma and evaluated the effect of statins and ezetimibe on PCSK9 in vivo and in vitro. In 254 normal subjects, the mean plasma PCSK9 was 89 +/- 32 ng/ml. PCSK9 levels correlated with plasma cholesterol, LDL-C, triglycerides, fasting glucose, age and body mass index. Sequencing PCSK9 from subjects at the extremes of plasma distribution revealed new variants. In 200 hypercholesterolemic patients, circulating PCSK9 was higher than in controls, increased with increasing statin dose, and further increased when ezetimibe was added. However, ezetimibe treatment of HepG2 (hepatocytes) and Caco-2 (enterocytes) cells caused a slight increase in PCSK9 and NPC1L1 mRNA, but no significant rise in PCSK9 protein secretion, suggesting that these transformed cells are not ideal model cell lines.