Our aims were i) to determine which lipids colocalize with newly synthesized apolipoprotein (apo) B in the lumen of the rough endoplasmic reticulum (RER), and thus may play a role in the stabilization and/or translocation of this protein; and ii) to determine the intracellular sites of assembly of lipids into very low density lipoprotein (VLDL). In order to do this, we have developed a new method for the separation of ER-derived microsomes on self-generated gradients of iodixanol. Rabbit liver microsomes were resolved into two broad peaks, the lighter peak contained smooth vesicles and the heavier peak contained rough vesicles. Each peak was collected in a number of subfractions. A single gradient thus separates the initial events in the secretion process (RER fractions), from later events (smooth endoplasmic reticulum (SER) fractions). The microsomal fractions were separated into membranes and lumenal contents, and the mass of apoB and VLDL lipids determined by ELISA or high performance thin-layer chromatography, respectively. The biosynthetic relationships of apoB and lipids were investigated, in timed or chase experiments, by incubation of isolated rabbit hepatocytes with radiolabeled precursors of apoB or lipids, followed by isolation and analysis of the microsomal fractions. The results indicate that very small amounts of triacylglycerol, cholesterol, and cholesteryl ester co-localize with apoB into the lumen of the RER. The bulk of the VLDL lipids were in the lumen of the SER. However, some newly synthesized triacyl-glycerol, phospholipid, cholesterol, and cholesteryl ester were also transferred to the lumen of the RER and were chased into the SER lumen. Double-labeling experiments showed that cholesteryl ester produced from newly synthesized cholesterol (labeled with [3H]mevalonate and [14C]oleate) was almost exclusively present in the RER, while cholesteryl ester in the SER was labeled only with [14C]oleate. Thus, distinct intracellular lipid-pools may be involved at different stages in the assembly of VLDL.