We have previously shown that different fatty acids (FA) influence the efficiency of synthesis and secretion of triacylglycerol (TG) and phospholipid (PL) in a newborn swine enterocyte cell line (IPEC-1) (Am J Physiol 272:G935, 1997; Am J Physiol 280:G1137, 2001). The most striking effects were produced by stearic acid (SA, 18:0), which modestly affected TG and PL synthesis, but reduced TG and PL secretion, and by eicosapentaenoic acid (EPA, 20:5), which reduced TG and PL synthesis and TG secretion, relative to oleic acid (OA, 18:1). To define the cellular mechanism of these effects, differentiated IPEC-1 cells were incubated for 24 hrs with OA, SA, or EPA and [³H]glycerol. Endoplasmic reticulum (ER) and Golgi (G) vesicles were prepared on a sucrose gradient. Organelle content of labeled lipids was measured, as well as apo B and A-I protein mass. The G/ER ratio was also determined for both TG and PL DPM and apolipoprotein mass. Relative to OA, SA did not impair ER TG synthesis, but reduced movement of labeled TG from ER to G. EPA impaired both ER TG synthesis and movement of labeled TG from ER to G. PL generally followed the same pattern, except ER synthesis of PL was relatively unaffected by EPA. Carbonate treatment demonstrated decreased partitioning of labeled lipid from membrane to lumen in cells treated with EPA. Organelle apo B and A-I content generally demonstrated opposite patterns after SA and EPA incubation. We conclude that SA and EPA adversely influence immature enterocyte ER to G lipid trafficking, as compared to OA. Furthermore, EPA appears to inhibit ER lipid synthesis and transfer of membrane lipid to luminal particles. Regulation of apo A-I ER to Golgi trafficking appears to be independent of that of apo B.