Immunoaffinity chromatography on a column of rabbit IgG anti-rat apolipoprotein (apo) A-I covalently bonded to agarose was used to isolate nascent high-density lipoprotein (nHDL) from recirculated perfusates of rat livers. After passage through the affinity column, the bound material was eluted with sodium thiocyanate and analyzed for apolipoproteins and lipids. The protein content was 52% and the lipid composition was 37% triglyceride, 40% phospholipid, and 23% cholesterol. Apoliproteins E and A-I each comprised approximately one third of the total, and very little apo B was detectable as judged by SDS-PAGE analysis. The affinity-isolated particles were therefore similar in composition to the major apo A-I:apo E-rich subfraction of nHDL isolated by ultracentrifugation in earlier work. It is concluded that the apo E in this class of nHDL (containing both apo E and apo A-I) is present in the secreted particle and is not a consequence of a loss of apo E from very-low-density lipoprotein (VLDL) during ultracentrifugation. The high triglyceride content in the virtual absence of apo B confirms and extends previous analyses and reinforces the conclusion that nHDL particles are enriched in triglyceride compared to plasma HDL. The inclusion of 4% albumin in the perfusion medium did not significantly change the total triglyceride output of 115 μg/g liver/h, but it decreased the triglyceride output isolated by anti-apo A-I affinity chromatography from 3.2 to 0.48 μg/g liver/h. The addition of oleic acid complexed to albumin increased the total triglyceride output by 70% and that associated with the immunoaffinity column increased from 0.48 to 2.7 μg/g liver/h. Under these conditions, the triglyceride content reached a value of 62% of the total lipid. These results indicate that the triglyceride content of apo A-I-associated nHDL responds to changes in hepatic triglyceride output in much the same way as that of nascent hepatic VLDL. The output of immunoaffinity-isolated apoproteins was unaffected by the presence of albumin in the perfusion medium, but the addition of oleate caused a 59% decrease in the amount of apo A-I and a 53% decrease in apo C, with no significant change in apo A-IV or apo E. We conclude that when the supply of free fatty acids to the liver is inceased, the entire range of nascent lipoproteins, from large VLDL to small HDL particles, become enriched in triglyceride.