The aim of this study was to investigate the types and characteristics of chylomicron precursors in the lumen of the secretory compartment of rabbit enterocytes. Luminal contents were separated into density subfractions in two continuous self-generating gradients of different density profiles. In enterocytes from rabbits fed a low fat diet, newly synthesized and immunodetectable apoB48 was only in the subfraction of density similar to high density lipoprotein (dense particles); the luminal triacylglycerol (TAG) content was low and only in the subfraction of density similar to that of chylomicrons/very low density lipoproteins (light particles). After feeding fat, newly synthesized, and immunodetectable apoB48 was in both dense (phospholipid-rich) and light (TAG-rich) particles. Luminal TAG mass and synthesis increased after fat feeding and was only in light particles. Pulse-chase experiments showed that the luminal-radiolabeled apoB48 lost from the dense particles was recovered in the light particles and the secreted chylomicrons. All of the light particle lipids (mass and newly synthesized) co-immunoprecipitated with apoB48. However, in the dense particles, there was a preferential co-precipitation of the preexisting rather than newly synthesized phospholipid. Assembly of apoB48-containing TAG-enriched lipoproteins is therefore a two-step process. The first step produces dense apoB48 phospholipid-rich particles, which accumulate in the smooth endoplasmic reticulum lumen. In the second step, these dense particles rapidly acquire the bulk of the TAG and additional phospholipid in a single and rapid step.