To investigate the assembly pathway for hepatic very low density lipoproteins (VLDL), nascent lipoproteins were recovered from a purified, intact rough endoplasmic reticulum (ER) fraction isolated from rat liver. Two fractions were recovered by ultracentrifugation. Particles isolated at d 1.006 g/ml were triglyceride-rich particles containing apolipoprotein (apo)B-100 or apoB-48, and apoE with very small amounts of apoA-I. Compared with VLDL recovered from the Golgi apparatus, the particles from the rough ER had less triglyceride, but more cholesteryl ester and phospholipid. The second class of particles isolated between d 1.006 and 1.210 g/ml were phospholipid-rich and contained apoB-48, apoE, and apoA-I. ApoB-100 was a minor component. Radioisotope incorporation studies utilizing [3H]leucine revealed differential rates of labeling of the apoproteins in these two lipoprotein fractions. ApoB-100 and apoE followed similar patterns in both fractions with peak incorporation occurring within 15 min of isotope injection. Incorporation of [3H]leucine into apoB-48 in the dense fraction peaked within 15 min of isotope administration, but peak incorporation in the d 1.006 g/ml fraction did not occur until approximately 30 min after injection. We propose that the two lipoprotein fractions recovered from the rough ER are intermediates in the assembly of VLDL by the liver. Comparison of the composition of these two particles with that of Golgi VLDL supports the sequential assembly of VLDL by the liver. Furthermore, we propose that the initial steps in the assembly of apoB-100- and apoB-48-containing lipoproteins are different with nascent apoB-100-containing particles being formed through the cotranslational association of this apoprotein with lipid while nascent apoB-48-containing VLDL are formed in the rough ER through a two-step process.