Rat HDL containing [stearic acid-14C, (methyl-3H)choline]sphingomyelin was prepared by incubating labelled sphingomyelin liposomes with serum. HDL was then separated by ultracentrifugation and purified by gel-filtration chromatography. The maximum transfer was reached when 1.5 microliter sphingomyelin was incubated in the presence of 1 ml of serum at 37 degrees C for 1 h. When transfer was limited to a 5-7% increase in HDL mass, no significant change was observed in the HDL electrophoretic pattern, and rats could therefore be injected with this type of HDL under physiological conditions. Plasma radioactivity decay was followed for 24 h, and the recovery of both isotopes in 11 tissues was studied 24 h after the injection. The decay in plasma of both isotopes followed three exponential phases. During the first two phases, both isotopes disappeared with the same velocity (t1/2 = 12.8 and 98-105 min for the first and second phases, respectively). 10 h after injection, 3H had disappeared more slowly than 14C (t1/2 = 862 and 502 min for 3H and 14C, respectively) and 24 h after injection, only 1.5% of 14C and 2.5% of 3H remained in the plasma. This radioactivity was located mainly in HDL (80-85% for 3H and 14C), with a 3H/14C ratio close to that of injected sphingomyelin, and in VLDL, with the same isotopic ratio as that of liver lipids. Some 3H was associated with non-lipoprotein proteins. 17.5% of 3H and 23.4% of 14C were recovered in the liver, 1.6% of each isotope in erythrocytes, and 1.4% of 3H and 0.6% of 14C in kidney. Less than 1% of each isotope was recovered in each of the other tissues. Phosphatidylcholine was the lipid most labelled, and in several tissues sphingomyelin had a 3H/14C ratio close to that of injected sphingomyelin, showing an uptake without prior hydrolysis.