—When cultured cells are exposed to plasma, the initial acceptors of unesterified cholesterol are small lipid-poor apolipoprotein A-I (apoA-I)–containing high density lipoproteins (HDLs) with pre-β electrophoretic mobility. These are converted by lecithin:cholesterol acyltransferase into larger spheroidal cholesteryl ester–rich HDLs with α mobility. To study the determinants of the concentration of small pre-β HDLs in tissue fluids, we collected prenodal peripheral lymph from 34 fasted normal men. By crossed immunoelectrophoresis, the concentration of pre-β HDLs in lymph averaged 20% of that in plasma. On multiple regression analysis, pre-β apoA-I concentration in lymph was directly related to pre-β apoA-I concentration in plasma and independently to α apoA-I concentration in lymph. Similar results were obtained when the same apoA-I–containing particles were quantified by size exclusion chromatography. Lymph pre-β apoA-I concentration was low in a subject with familial lecithin:cholesterol acyltransferase deficiency, despite a normal plasma pre-β apoA-I concentration, but was normal in a subject with familial lipoprotein lipase deficiency. These results suggest that the concentration of small pre-β HDLs in human tissue fluids is determined only in part by the transfer of pre-β HDLs across capillary endothelium from plasma. Local production, by remodeling of spheroidal α HDLs in tissue fluids, may be equally important. Lipolysis of triglyceride-rich lipoproteins by lipoprotein lipase appears to have little effect.