A common feature of cystic fibrosis (CF) is the functional derangement of the exocrine pancreas, which affects output of pancreatic lipase. This condition results in severe dietary malabsorption due to the poor hydrolysis of triacylglycerol (TG) in the lumen of the small intestine. Despite the benefits of pancreatic enzyme supplements, persistent intestinal fat malabsorption characterizes patients with CF. The aim of the current investigation was to determine whether defects in the intracellular phase of lipid transport occur in this pathophysiology in addition to the known disturbed digestive processes. Our hypothesis was tested by incubating intestinal biopsies from 6 CF and 6 healthy subjects with radiolabelled lipid and protein precursors. Lipid esterification and secretion were markedly decreased by 22-31% and 38-42%, respectively, in CF as noted by the low incorporation of (¹⁴C)-palmitic acid into TGs, phospholipids and cholesteryl esters in patients duodenal explants and culture media compared with controls (100%). Accordingly, the output of TG-rich lipoproteins was substantially reduced (p<0.05) and a similar trend was observed for high-density lipoproteins. Since intestinal lipoprotein assembly/secretion shows an absolute requirement for apolipoprotein (apo) B-48, radioactive labeling experiments were performed and they demonstrated a significantly (p<0.05) diminished synthesis of apo B-48 (40%) and apo A-I (30%). Given the critical role of microsomal triglyceride transfer protein in the formation of apo B-containing lipoproteins, its activity was determined and not found to be altered in CF intestinal tissue. Taken together, these results suggest that CF malabsorption may also be caused by defects in mucosal mechanisms leading to abnormal lipoprotein delivery into the blood circulation.