Under lipid-poor conditions, most newly synthesized apolipoprotein B100 (apoB) undergoes rapid degradation in Hep G2 cells such that only a small fraction of newly synthesized apoB is actually secreted. Addition of oleate to Hep G2 culture medium stimulates apoB secretion by a post-translational mechanism. In the current studies we have explored oleate-stimulation of apoB secretion by using calpain inhibitor I, N-acetyl-leucyl-leucyl-norleucinal (ALLN), a compound that inhibits the intracellular degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and the T cell receptor alpha subunit. Preincubation of Hep G2 cells with ALLN (40 micrograms/ml) for 1 h markedly inhibited degradation of newly synthesized apoB. Whereas only 32% of newly labeled apoB remained intact (cells+medium) in control cells after a 10-min pulse with [3H]leucine followed by a 60-min chase, 84% of labeled apoB was intact in ALLN-treated cells. However, most of the ALLN-protected apoB remained intracellular, as ALLN did not stimulate the rate of apoB secretion over the control rate (12 versus 9.2%). Although secretion of apoB was not accelerated, the protection afforded by ALLN continued for several hours, and labeled apoB continued to be secreted over 3 h of chase after which secretion ceased. The protection afforded by ALLN resulted in 37% of labeled apoB secreted by 3 h compared to 15% in control cells. In contrast, simultaneous treatment of cells with ALLN and oleate both accelerated and increased total apoB secretion, such that 36% of initially labeled apoB was recovered in the medium by 60 min and 71% of labeled apoB was secreted by 180 min of chase. These data show that ALLN and oleate affect apoB metabolism by different mechanisms. Although ALLN can protect nascent apoB from rapid early intracellular degradation, it does not accelerate apoB secretion. In contrast, although our results can not rule out the possibility that oleate may directly inhibit proteolysis of apoB, oleate appears to protect apoB mainly by facilitating transport of apoB out of a protease-containing compartment associated with the endoplasmic reticulum.