Nascent high density lipoprotein (HDL) (1.063 less than d less than 1.21 g/ml) was isolated from recirculating rat liver perfusates and separated by heparin-Sepharose chromatography into a nonretained fraction (NR) and a fraction (R) that eluted with 0.5 M NaCl. Fractions NR and R contained 70% and 30% of the nascent HDL protein, respectively. ApoB-containing particles were removed from fraction R by chromatography on concanavalin A. The protein composition of fractions NR and R was 40% and 29%, respectively. Fraction NR contained 25% apoA-I, 11% apoA-IV, 24% apoE, and 38% apoC. Fraction R contained primarily apoE (81% of total protein). The lipid composition of NR and R, respectively, was: triglyceride 44% and 26%, phospholipid 41% and 57%, cholesterol 8% and 13%, and cholesteryl ester 7% and 4%. Fractions NR and R had molecular weights of 400,000 and 860,000, respectively, as calculated from the Stokes radius. Negative staining electron microscopy indicated that both fractions consisted mainly of spherical particles (260-280 A) but some stacked disks were seen in fraction R. Livers perfused by the single-pass technique produced fractions NR and R in the same ratio as livers perfused by recirculation. The apolipoprotein compositions were similar to those in the recirculating perfusion; however, both fractions NR and R had more triglyceride (greater than 50% of total lipid). An HDL fraction was also isolated from liver perfusates by a combination of molecular sieve and heparin-Sepharose affinity chromatography. This HDL contained triglyceride but no apoB, indicating that triglyceride-rich HDL particles are not an artifact of ultracentrifugation.(ABSTRACT TRUNCATED AT 250 WORDS)