The objective of this study was to assess the relative long-term effects of linoleic (cis, cis 18:2), linolelaidic (trans, trans 18:2), and palmitic (16:0) acids on hepatic lipoprotein production in HepG2 cells. All fatty acids increased the mass of triglycerides (TG) in the medium and the incorporation of [(3)H]-glycerol into secreted TG; the increase was more pronounced with linoleic acid than with linolelaidic and palmitic acids. The net accumulation in the medium of apolipoprotein (apo) A-I was not affected by the fatty acids tested and moderate changes in that of apoB resulted in apoB/apoA-I mass ratios of 1.05, 1.27 and 0.86 with linoleic, linolelaidic and palmitic acids, respectively. The incorporation of [(14)C]-acetate into cellular plus secreted total sterols was 9.1%, 33.6% and 17.4% of total [(14)C]-labeled lipids with linoleic, linolelaidic and palmitic acids, respectively. Relative to linoleic acid, palmitic acid, and to a greater extent (P < 0.05) linolelaidic acid, increased the secretion and cellular accumulation of [(14)C]-labeled free cholesterol (FC) and cholesteryl esters and decreased those of TG and phospholipids (PL). Compared with linoleic acid, linolelaidic acid increased LDL-cholesterol (C) and HDL-C by 154% (P < 0.001) and 50% (P = 0.016), respectively, whereas palmitic acid increased LDL-C by 17% (P > 0.1) and did not affect HDL-C. The LDL-C to HDL-C ratios were 0.70, 1.18 and 0.96 with linoleic, linolelaidic and palmitic acids, respectively. These differences were not due to altered LDL receptor activity. The PL to C ratios of HDL particles were 1.61, 0.40 and 0.77 with linoleic acid, linolelaidic acid and palmitic acid, respectively. These results suggest that relative to cis polyunsaturated and saturated fatty acids, trans PUFA more adversely affect the concentration and composition of apoA-I- and apoB-containing lipoproteins secreted by HepG2 cells.