Apolipoprotein (apo) B is an obligatory component of triglyceride-rich lipoproteins. In the rare autosomal recessive disorder abetalipoproteinemia (ABL), no triglyceride-rich lipoproteins are secreted. Mutations in the gene encoding the 97-kDa subunit of a microsomal triglyceride transfer protein (MTP) cause ABL (Sharp, D., Blinderman, L., Combs, K. A., Klenzle, B., Ricci, B., Wager-Smith, K., Gil, C. M., Turck, C. W., Bouma, M. E., Rader, D. J., Aggerbeck, L. P., Gregg, R. E., Gordon, D. A., and Wetterau, J. R. (1993) Nature 365, 65-69; Shoulders, C. C., Brett, D. J., Bayliss, J. D., Narcisi, T. M., Jarmuz, A., Grantham, T. T., Leoni, P. R. D., Bhattacharya, S., Pease, R. J., Cullen, P. M., Levi, S., Byfield, P. G. H., Purkiss, P., and Scott, J. (1993) Hum. Mol. Genet. 2, 2109-2116). Here we have examined whether MTP is both necessary and sufficient to mediate the secretion of apoB-containing lipoproteins from cells that do not normally express either of these proteins. Carboxyl-terminal truncated forms of apoB, apoB17, and apoB41 on the centile system were expressed in COS-1 cells. ApoB17 was secreted whereas apoB41 was unable to traverse the secretory pathway. Cotransfection of apoB41 and MTP promoted the secretion of apoB41 as a buoyant lipoprotein particle with a modal density of 1.15 g/ml. When cotransfected COS-1 cells were cultured under conditions that increase the secretion of apoB100 from HepG2 cells, secretion of apoB41 was similarly increased. N-Acetyl-leucyl-leucyl-norleucinal (ALLN), a calpain I inhibitor, abolished intracellular degradation of apoB41 and increased secretion 2.5-fold. Oleate, a substrate for triglyceride synthesis, reduced degradation from 50 to 19% and increased secretion by 2.5-fold. The effects of ALLN and oleate were additive. We conclude that the secretion of apoB from COS-1 cells cotransfected with apoB and MTP is determined by the competitive processes of lipoprotein assembly and intracellular degradation in the endoplasmic reticulum and that MTP is the only tissue-specific component, other than apoB, required for the secretion of apoB-containing lipoproteins.