We have shown that non-hepatic Chinese hamster ovary cells (CHO) have a specific inability to translocate and secrete apolipoprotein B (apoB), leading to its complete degradation in the endoplasmic reticulum (Thrift, R. N., Drisko, J., Dueland, S., Trawick, J. D., and Davis, R. A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 9161-9165). To gain an understanding why a protein having no predictable trans-membrane sequences can be stably integrated into the endoplasmic reticulum, we determined the topography and metabolic fate of apoB in both CHO cells and human hepatoma cells (HepG2). Using epitope-specific antibodies, we show that in microsomes from both cell types, apoB assumes a trans-membrane orientation having 69 kDa of its N terminus in the lumen and the remaining portion of the C terminus residing on the cytoplasmic surface. In both cell types, proteolytic cleavage of the translocation arrested apoB by a process that can be blocked by acetyl-leucine, leucine, norleucal, produces an 85-kDa N-terminal fragment that resumes translocation and is secreted. This same N-terminal 85-kDa fragment is also found in human plasma. These results show that sequences residing outside of the membrane spanning domain can block translocation. Moreover, our data provide compelling evidence showing that apoB undergoes an unusual transient, translocation arrest, that serves as an entrance into the intracellular degradative pathway regulating its secretion.