Hyperlipidemia arises from a disturbance in the balance between production and degradation of lipoprotein particles. Variation in the secretion of human apolipoprotein B (apoB), the major protein component of triglyceride-rich lipoproteins, directly affects this homeostasis. Naturally occurring apoB signal peptide variants (associated with hypertriglyceridemia, altered postprandial lipid metabolism, or atherosclerosis) were investigated for their ability to direct transit through the secretion pathway. Three apoB signal peptide isoforms were fused to the secretory protein, invertase, and expressed in yeast. A deletion or insertion in the hydrophobic core of the signal peptide mediated inefficient translocation into the endoplasmic reticulum and was secretion-defective, relative to the common 27-residue isoform. Additionally, the insertion apoB isoform was observed in yeast to confer a defect in export from the endoplasmic reticulum. Secretion of the apoB signal peptide-invertase fusions responded positively to an inhibitor of calpain type I proteases. These observations suggest that the apoB signal peptide plays a role in determining the levels of apoB degradation and secretion and, thus, hyperlipidemia.