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Evidence gradually accumulated by moving boundary electrophoresis (1-4), chemical fractionation, (5-7) using method of Cohn and his associates (8) biliary cirrhotic ACD 2 plasma and 9 ml. of normal ACD2 plasma were dialyzed against several changes of citrate-sodium chloride reagent8 for 48 hours at 0°C. Densities of the dialyzed solutions were adjusted to approximately 1.065 with 4.0 M NaCl and distilled water until a final volume of 11.0 ml. was obtained for the biliary cirrhotic and 13.0 ml. for the normal plasma. Nineml. aliquots of each were placed in 13 X 85 mm. plastic centrifuge tubes and ultracentrifuged at 30,000 rpm (80,000 G) for 18 to 20 hours at 50 C. The top 3.0 cm. of solution were separated with a tube-slicing device. From the upper and lower cuts containing low and high density lipoproteins, respectively, 100 X samples were removed for determination of solution density. The upper cut of the biliary cirrhotic plasma was diluted to 6.0 ml. with 2.3 M NaCl and from the normal plasma with 2.0 M NaCl. An aliquot of 4.5 ml. was removed from each into 13 X 85 mm. plastic tubes for density gradient studies (described presently); the remainder was dialyzed together with both lower cuts (high density lipoproteins) against 0.15 M NaCl for 24 hours at 0°C. The dialyzed solutions were then analyzed for lipids, nitrogen, and dry weight.A linear density gradient as described by LinderstromLang and Lanz (13,14) and Anfinsen (15) was made of the diluted upper cuts as follows: 4.5 ml. 0.1 M NaCl were layered over the 4.5-ml. aliquot of low density lipoproteins that had been previously placed in a 13 X 85 mm. plastic tube. A simply-constructed wire stirrer with a spiral button tip was gently inserted to the interface of the two solutions, from which point gentle vertical strokes of increasing 0.75 cm. distances were made, keeping the plane of the spiral tip as level as possible. The manipulation usually required four strokes each down and up from the interface. The tubes were then ultracentrifuged at 30,000 rpm for 18 to 20 hours at 5°C. Lipoproteins concentrate in such gradients at layers where the lipoprotein density is equal to the density of the sodium chloride solution. The resulting gradients were partitioned into 8 or 9 cuts, 0.75 cm. each, either by slicing with a tube-slicing device or by aspirating with a finetipped pipette. From the volume of each cut (approximately 1.0 ml.) a 100 X sample was removed for determination of solution density. To the remainder of each cut was added an equal volume of 0.15 M NaCl; each 2ACD (acid citrate-dextrose) is the special anticoagulant solution used when fractionating plasma proteins by the Cohn method (8).8 Citrate-sodium chloride reagent consisted of 0.02 M trisodium citrate and 0.07 M sodium chloride.133