The evidence is strong that biological functions contained in high-density lipoproteins (HDL) are antiatherogenic. These functions may track with HDL cholesterol or apolipoprotein A1 concentration to explain the strongly inverse risk curve for cardiovascular disease. Moreover, there are harmful as well as protective HDL subspecies in regard to cardiovascular disease, which could be responsible for paradoxical responses to HDL-directed treatments. Recent metabolic studies show that apolipoprotein A1–containing HDL is secreted into the circulation as mostly spherical cholesterol ester–rich lipoproteins that span the HDL size range. Most of the flux of apolipoprotein A1 HDL into and out of the circulation occurs in these spherical cholesterol-replete particles. Discoidal cholesterol-poor HDL comprises a minority of HDL secretion. We propose that much cholesterol in reverse cholesterol transport enters and exits medium and large size HDL without changing a size category, and its flux may be estimated provisionally from holoparticle clearance of cholesterol ester–rich HDL. An accurate framework for metabolism of HDL is essential to finding steady-state biomarkers that reflect HDL function in vivo. Whereas cholesterol efflux from cells to mainly discoidal HDL, mediated by ABCA1 (ATP-binding cassette transporter ABCA1), predicts cardiovascular disease, cholesterol transfers to spherical HDL also can be measured and may be relevant to protection against atherosclerosis. We propose several investigative paths on which human HDL biology may be investigated leading to convenient biomarkers of HDL quality and function having potential not only to improve risk prediction but also to more accurately target drug treatments.