We have used a panel of anti-rabbit apolipoprotein B monoclonal antibodies in a competitive ELISA to probe the availability of apoB in rough endoplasmic reticulum, smooth endoplasmic reticulum, cis-enriched Golgi, and trans-enriched Golgi fractions from rabbit liver. The ability of each subcellular fraction to inhibit binding of monoclonal antibody to immobilized low density lipoprotein (LDL)-apoB was determined and compared with the expected inhibition based on the apoB content of the fraction. The vesicles remained closed during ELISA, demonstrated by monitoring loss of radiolabeled secretory proteins from the lumen and by measuring leakage of albumin from the vesicles. In control experiments, vesicles were permeabilized using 0.4% taurocholate. All epitopes of apoB were fully expressed in closed trans-Golgi vesicles, indicating that the membrane-bound apoB is at the cytosolic side of this fraction. In the smooth endoplasmic reticulum the epitopes were expressed between 55 and 70%, suggesting that the two pools of apoB may exist in these membranes. These results suggest that newly synthesized apoB has two possible fates. It may be incorporated into the cytosolic side of the endoplasmic reticulum from where it moves to the cytosolic side of the Golgi membrane, or newly synthesized apoB may be translocated to the lumenal surface of the endoplasmic reticulum membrane followed by assembly with lipids for secretion.