Nondenaturing gradient gel analysis of high density lipoproteins (HDL, d 1.063-1.235 g/ml) isolated from Hep G2 24-hr, serum-free conditioned media shows four distinct, reproducible particle subclasses I, II, III, and IV with apparent Stokes' diameters of 13.3, 12.0, 9.5, and 7.4 nm, respectively. Fractions enriched in lipoproteins from each of these subclasses were isolated by either density gradient ultracentrifugation or gel filtration chromatography and characterized. Size and morphology of the isolated subclasses agreed well regardless of isolation procedure. Electron microscopy revealed subclasses I, II, and III to be disc-shaped, and subclass IV to be spherical. The discoidal subclasses were poor in cholesteryl ester and rich in phospholipid and unesterified cholesterol. The larger-sized subclass I particles were enriched in apolipoprotein (apo) E while subclasses II and III had decreasing amounts of apoE and increasing amounts of apoA-I and A-II. The spherical subclass IV particles contained a higher percentage of protein and had a higher ratio of cholesteryl ester to unesterified cholesterol than that found in the other subclasses. Subclass IV contained predominantly apoA-I. The subclasses isolated from Hep G2 HDL appear to share many similarities with those isolated from patients with lecithin:cholesterol acyltransferase deficiency and are therefore potentially useful in examining the transformation of nascent HDL particles to mature circulating plasma forms.