High density lipoproteins (HDL) were isolated by a chromatographic procedure and subsequently fractionated on a DEAE cellulose (DE-52) column. Four fractions were separated and analyzed for lipid and protein composition and molecular weight. During ion exchange chromatography, one of the four fractions consistently coincided with lecithin:cholesterol acyltransferase (LCAT) activity. When the HDL fractions were incubated with highly purified LCAT preparations, the LCAT activity showed a dependence on unesterified cholesterol concentrations. The HDL subfraction eluting at the highest ionic strength was found to be the best substrate for LCAT. This subfraction exhibited apoprotein and lipid composition similar to HDL3 and contained 31% of the total apoprotein D present in all the subfractions. A positive correlation was found between LCAT activity and the cholesteryl ester/unesterified cholesterol ratio, and a negative correlation was found between LCAT substrate potential and apparent molecular weight of the HDL subfractions when these subfractions were incubated with LCAT. No correlation was apparent between LCAT activity, and the phospholipid/unesterified cholesterol ratio or with the apoA-I/apoA-II ratio.U