Fish oils are rich in omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), predominantly 20:5n-3 and 22:6n-3, whereas vegetable oils contain abundant C18-PUFA, predominantly 18:3n-3 or 18:2n-6. We hypothesized that replacement of fish oils with vegetable oils would increase the oxidative stability of fish lipids. Here we have used the long established and easily cultivated FHM cell line derived from the freshwater fish species fathead minnow (Pimephales promelas) to test this hypothesis. The FHM cells were readily able to synthesize 20:5n-3 and 24:6n-3 from 18:3n-3 but 22:6n-3 synthesis was negligible. Also, they were readily able to synthesize 20:3n-6 from 18:2n-6 but 20:4n-6 synthesis was negligible. Mitochondrial β-oxidation was greatest for 18:3n-3 and 20:5n-3 and the rates for 16:0, 18:2n-6, 22:6n-3 and 18:1n-9 were significantly lower. Fatty acid incorporation was predominantly into phospholipids (79-97%) with very little incorporation into neutral lipids. Increasing the fatty acid concentration in the growth medium substantially increased the concentrations of 18:3n-3 and 18:2n-6 in the cell phospholipids but this was not the case for 20:5n-3 or 22:6n-3. When they were subjected to oxidative stress, the FHM cells supplemented with either 20:5n-3 or 22:6n-3 (as compared with 18:3n-3 or saturated fatty acids) exhibited significantly higher levels of thiobarbituric reactive substances (TBARS) indicating higher levels of lipid peroxidation. The results are discussed in relation to the effects of fatty acid unsaturation on the oxidative stability of cellular lipids and the implications for sustainable aquaculture.