Isolated rabbit hepatocytes were incubated with [35S]methionine to label intracellular pools of apolipoprotein B (apo-B). The cells were then reincubated with an excess of unlabelled methionine in the presence of oleate or protease inhibitors and the intracellular sites of accumulation of radiolabelled apo-B and the mass of apo-B were determined by isolation and analysis of subcellular fractions. Oleate or inhibitors of metalloproteases (o-phenanthroline), serine proteases (aprotinin), serine/cysteine proteases (leupeptin) or cysteins proteases (calpain inhibitor I; ALLN) but not aspartate proteases (pepstatin) resulted in inhibition of the cellular degradation of apo-B. The effect of o-phenanthroline was reversed by the addition of zinc ions. Oleate, o-phenanthroline and leupeptin also stimulated secretion of radiolabelled apo-B; the effects of the inhibitors and oleate were additive, suggesting that they could act via different mechanisms. o-Phenanthroline caused accumulation of apo-B in the rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER) membranes; leupeptin caused accumulation of apo-B in the SER and cis-Golgi membranes, and ALLN and aprotinin caused accumulation of apo-B in the trans-Golgi membranes. These results suggest that intracellular degradation of apo-B occurs in the endoplasmic reticulum and in the trans-Golgi membranes and involves different proteases. Apo-B that accumulates in the ER membrane can be diverted into the lumen for secretion; however, apo-B that accumulates in the trans-Golgi membrane is irretrievably diverted from secretion.