The effects of the hypolipidemic fatty acid analogue tetradecylthioacetic acid (TTA) on synthesis and secretion of lipoproteins in CaCo-2 cells were studied. Radiolabeled tetradecylthioacetic acid was absorbed and metabolized as efficiently as oleic acid, although a discrepancy in the metabolic fate was evident. Whereas tetradecylthioacetic acid was incorporated into cell-associated triacylglycerol to the same extent as normal fatty acids (e.g., oleic acid and palmitic acid), the amount of triacylglycerol secreted from cells incubated with tetradecylthioacetic acid was 8 to 10 times lower than the amount secreted from cells incubated with palmitic acid and oleic acid, respectively. On the other hand, there was an enhanced incorporation of tetradecylthioacetic acid into cell-associated and secreted phospholipids. Despite incorporation of tetradecylthioacetic acid into cellular triacylglycerol, unlike oleic acid, tetradecylthioacetic acid did not stimulate production of triacylglycerol-rich particles. Ultracentrifugation of basolateral media from cells incubated with tetradecylthioacetic acid revealed low amounts of triacylglycerol in the triacylglycerol-rich fraction (p < 1.006 g/ml), suggesting secretion of lipoproteins with a higher density than chylomicrons. However, the present study shows that the stimulated triacylglycerol secretion caused by oleic acid was inhibited in the presence of TTA. The decreased rate of triacylglycerol secretion from these cells was not accompanied by a stimulation of fatty acid oxidation. Based on these findings, we therefore suggest that tetradecylthioacetic acid mainly affects secretion of lipoproteins in CaCo-2 cells.