The mechanisms for packaging large quantities of neutral lipids into apolipoprotein (apo) B-containing lipoproteins (chylomicrons or VLDL) are incompletely understood. However, several lines of evidence have suggested that the addition of core lipids to apoB to form a lipoprotein particle within the endoplasmic reticulum (ER) may involve two steps: first, the addition of small amounts of core lipids to membrane-bound apoB, generating a lipid-poor, small apoB-containing particle, and second, the fusion of that particle with a larger, independently formed triglyceride-rich and apoB-free "lipid particle." We sought to test this two-step hypothesis of apoB core lipidation by using electron microscopy to compare chylomicron assembly in mice that are genetically deficient in the ability to synthesize apoB in the intestine to control mice. In 19-day gestational mice (fasting setting) that were deficient in intestinal apoB synthesis, chylomicron-sized lipid particles in the lumen of the enterocyte ER were even more abundant and were 2- to 3-fold larger than those in the enterocytes of normal control mice. However, there were fewer lipid-staining particles in the Golgi apparatus, and many fewer particles in the extracellular space, compared with normal control mice. In both types of newborn suckling mice, much larger lipid particles were assembled within the lumen of the ER. They were however, less abundant and rarely reached the Golgi apparatus in fatty enterocytes of intestines deficient in apoB synthesis. These observations provide in vivo evidence that chylomicron formation could involve the synthesis of apoB-free triglyceride-rich particles within the endoplasmic reticulum (ER) lumen, and that the transport of these lipid particles out of the ER to Golgi apparatus and interstitium is facilitated by the acquisition of apoB.