Dietary n-3 polyunsaturated fatty acids (PUFA) suppress the secretion of very low density lipoprotein (VLDL) directly when delivered to the liver in chylomicron remnants (CMR). The role of sterol regulatory element-binding proteins (SREBPs) and hepatic nuclear factor-4alpha (HNF-4alpha) in the regulation of this effect was investigated. Chylomicron remnant-like particles (CRLPs) containing triacylglycerol (TG) from palm (rich in saturated fatty acids (SFA)) or fish (rich in n-3 PUFA) oil were incubated with cultured rat hepatocytes (24h) and the expression of protein and mRNA for SREBP-1, SREBP-2 and HNF-4alpha, and levels of mRNA for their target genes were determined. SREBP-1 and -2 protein expression in the membrane and nuclear fractions was unaffected by either type of CRLPs. mRNA abundance for SREBP-1c and -2 was also unchanged by CRLP-treatment, as were levels of mRNA for target genes of SREBP-1, including steroyl CoA desaturase, acetyl CoA carboxylase, fatty acid synthase and ATP citrate lyase, and SREBP-2 (3-hydroxy-3-methylglutaryl CoA reductase). In contrast, HNF-4alpha protein and mRNA levels were significantly decreased by CRLPs enriched in n-3 PUFA, but not SFA, and the expression of mRNA for HNF-4alpha target genes, including HNF-1alpha, apolipoprotein B and the microsomal TG transfer protein, was also lowered by n-3 PUFA-, but not SFA-enriched CRLPs. These findings suggest that the direct suppression of VLDL secretion by dietary n-3 PUFA delivered to the liver in CMR is mediated via decreased expression of HNF-4alpha.