A method for the determination of the cholesterol content of human plasma high density lipoprotein (HDL) subfractions HDL2 and HDL3 is presented. The method is based on one preparative ultracentrifugal spin at density 1.125 to obtain HDL3 and heparin/manganese precipitation to obtain total HDL. From this spin in a fixed angle rotor at density 1.125 HDL3 is obtained as a bottom fraction in which cholesterol is determined. The total HDL cholesterol concentration is determined in the supernatant of the heparin/manganese precipitation of VLDL-free serum. HDL2 cholesterol is calculated as the difference between total HDL and HDL3 cholesterol. One of the advantages of this method is that falsely high HDL2 cholesterol levels due to incorporation of LP(a) containing B-protein in the HDL2 fraction is avoided. Some validation of the 1.125-spin is given. Results are presented from small samples of normal of both sexes as well as men with different kinds of hyperlipoproteinaemia.