A high resolution electrophoretic method has been developed to separate plasma high density lipoprotein (HDL) particles by size using 4-30% polyacrylamide agarose (PAA) gradient gels, Sudan black B staining, and laser densitometry. Fourteen distinct HDL bands were observed with HDL-1 being designated as the largest particle and HDL-14 as the smallest particle. HDL-1 was similar in size to ferritin (Stokes diameter 12.2 nm), HDL-8 to catalase (9.2 nm), and HDL-13 to lactate dehydrogenase (8.1 nm). HDL-1 to HDL-7 were found within the density range of HDL2b (d 1.063-1.10 g/ml), HDL-8 to HDL-10 within HDL2a (d 1.10-1.125 g/ml), and HDL-11 to HDL-14 within HDL3 (d 1.125-1.21 g/ml). On immunoblotting, apolipoprotein A-I (apoA-I) was found in all HDL bands examined, being most prominent in HDL-6, 11, and 12. ApoA-II was not detected in HDL bands 1-5, but was present in all other HDL bands and was most prominent in HDL-9. ApoE was detected mainly in HDL bands 1-7, and was observed in only trace amounts in other bands. Lp A-I isolated by immunoaffinity column chromatography from the plasma of five subjects contained five subspecies (HDL-5, 6, and 11-13), while Lp A-I/A-II also had five subspecies (HDL-8, 9, and 11-13) in these subjects. In normal subjects (n = 57) four or five HDL bands were generally observed, with HDL-9, 11, and 12 being the most frequently observed. Mean HDL particle score (method of sizing based on scanning densitometry, where low score indicates large size and high score indicates small size) was significantly correlated (P < 0.001) with the concentrations of HDL cholesterol (r = -0.796), HDL free cholesterol (r = -0.780), HDL cholesteryl ester (r = -0.683), HDL phospholipid (r = -0.663), HDL apoA-I (r = -0.577), and HDL protein (r = -0.459), but not with HDL triglyceride (r = 0.069). In addition, HDL particle score was significantly correlated (P < 0.05) with HDL total mass (r = -0.649), HDL free cholesterol content (% of total mass, r = -0.608), HDL triglyceride content (r = 0.415), HDL phospholipid content (r = -0.359), and HDL protein content (r = 0.295), but not with HDL cholesteryl ester content (r = -0.219) or HDL apoA-I content (r = 0.183).(ABSTRACT TRUNCATED AT 400 WORDS)