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Atlantic salmon were fed fish meal-based diets supplemented with either 100% fish oil (FO) or 100% rapeseed oil (RO) from an initial weight of 85 g to a final average weight of 280 g. The effects of these diets on the capacity of Atlantic salmon hepatocytes to elongate, desaturate, and esterify [1-14C] 18:1n-9 and the immediate substrates for the delta5 desaturase, [1-14C] 20:3 n-6 and [1-14C] 20:4n-3, were investigated. Radiolabeled 18:1n-9 was mainly esterified into cellular TAG, whereas the more polyunsaturated FA, [1-14C] 20:3n-6 and [1-14C] 20:4n-3, were primarily esterified into cellular PL. More of the elongation product, [1-14C] 20:1n-9, was produced from 18:1n-9 and more of the desaturation and elongation products, 22:5n-6 and 22:6n-3, were produced from [1-14C]20:3n-6 and [1-14C] 20:4n-3, respectively, in RO hepatocytes than in FO hepatocytes. Further, we studied whether increased addition of [1-14C]18:1n-9 to the hepatocyte culture media would affect the capacity of hepatocytes to oxidize 18:1n-9 to acid-soluble products and CO2. An increase in exogenous concentration of 18:1 n-9 from 7 to 100 microM resulted in a nearly twofold increase in the amount of 18:1n-9 that was oxidized. The conversion of 20:4n-3 and 20:3n-6 to the longer-chain 22:6n-3 and 22:5n-6 was enhanced by RO feeding in Atlantic salmon hepatocytes. The increased capacity of RO hepatocytes to produce 22:6n-3 was, however, not enough to achieve the levels found in FO hepatocytes. Our data further showed that there were no differences in the hepatocyte FA oxidation capacity and the lipid deposition of carcass and liver between the two groups.