The fatty acid analogue tetradecylthioacetic acid (TTA) has previously been shown to decrease triacylglycerol secretion in CaCo-2 cells (Gedde-Dahl et al., J. Lipid Res. 36 (1995) 535-543). The present study was designed to further elucidate the effect of TTA on lipoprotein production in CaCo-2 cells. TTA did not affect oleic acid-induced triacylglycerol synthesis, but it significantly decreased secretion of newly synthesized triacylglycerol when compared to cells incubated with oleic acid alone or oleic acid in combination with palmitic acid. In contrast, pulse-chase experiments showed no difference in the amount of labeled triacylglycerol secreted from cells exposed to either fatty acid combination during the chase period, indicating that TTA did not affect the secretory process in general. Cells incubated with TTA alone secreted triacylglycerol present at 1.025<rho<1.073 g/ml, corresponding to the low density lipoprotein/intermediate density lipoprotein density range. In contrast, cells supplemented with oleic acid or oleic acid in combination with TTA secreted triacylglycerol mainly in the very low density lipoprotein/chylomicron density range (rho<1.006 g/ml). Despite a marked decrease in triacylglycerol secretion, TTA treatment did not change secretion of apolipoprotein B nor the activity of microsomal triacylglycerol transfer protein (MTP) in the cells. Furthermore, the presence of TTA in cellular triacylglycerol had no effect on the ability of purified MTP to transfer triacylglycerol from donor to acceptor vesicles. Together, the above observations suggest that TTA interferes with other MTP-independent factors that regulate the intestinal lipoprotein secretion.