Cultured hepatocytes from rats fed a low fat, chow diet (LF) or diets rich in fish oil (FO, 20% v/w) or olive oil (OO, 20%, v/w) were used to determine how the intracellular metabolism and secretion of apolipoproteins (apo)B-100 and B-48 respond to in vivo and in vitro manipulations of fatty acid esterification, storage, secretion, and oxidation. Hepatocytes from the FO- and OO-fed rats had higher initial triacylglycerol (TAG) contents and higher rates of fatty acid oxidation and ketogenesis than hepatocytes from the LF-fed group. However, only in the cells from the FO-fed animals was there any decrease in the rate of TAG synthesis and in the secretion of VLDL TAG. Decreased secretion of TAG by the FO hepatocytes was accompanied by a decreased synthesis and degradation of apoB, particularly apoB-48, and a decreased secretion of apoB-48 VLDL. In all dietary groups a substantial proportion of the apoB-48 and apoB-100 secreted into the medium was associated with small, lipid-poor particles of density > 1.006. FO feeding had no effect on the amounts of apoB-48 and apoB-100 that appeared in this fraction. Dietary composition affected the channelling of exogenous oleate in the culture medium into the oxidative and esterification pathways. While exogenous fatty acid increased the secretion of VLDL TAG in the FO-fed group, VLDL TAG secretion remained lower than that observed in the hepatocytes from the LF- and OO-fed groups cultured under identical conditions. Exogenous oleate did not significantly increase the secretion of either newly-synthesized apoB-48 or apoB-100 by hepatocytes in either of the dietary groups. We conclude that, in rat liver, a decreased capacity to transport TAG out of the hepatocyte after consumption of a diet rich in fish oil is associated with a decreased synthesis and presecretory degradation of apoB-48, and with a decreased secretion of VLDL apoB-48.