The conditions for the separation of rat high density lipoproteins (HDL) in a single ultracentrifuge run are described. By this method six serum samples can be processed simultaneously. HDL is separated into two main fractions, one with apolipoprotein E and the other with apolipoprotein A-I as the major protein component. The apolipoprotein E-rich HDL contains a relatively high amount of phospholipid and unesterified cholesterol and therefore resembles HDL-1 or apolipoprotein E HDL as isolated by other methods. The other HDL fraction resembles HDL-2. The two HDL fractions appeared to be heterogeneous with respect to apolipoprotein composition. The HDL-1 consisted of particles with and without a low percentage of apolipoprotein A-I. The HDL-2 consisted of particles with a variable amount of apolipoprotein E and A-IV. During incubation of rat serum for 5 h at 37 degrees C in the presence of dithiobis(2-nitrobenzoic acid) (DTNB) a small shift of the HDL-2 peak to lower densities occurred. Incubation of the serum without DTNB led to a loss of cholesterol from the 'light' HDL-1 fractions and an increase in cholesterol ester in fractions at densities intermediate between those of HDL-1 and HDL-2 and in fractions at the densest part of the gradient.