Due to the scarcity of marine fish oil resources, the aquaculture industry is developing more efficient strategies for the utilization of dietary omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA). A better understanding of how fish utilize EPA and DHA, typically provided by fish oil, is needed. However, EPA and DHA have different physiological functions, may be metabolized and incorporated into tissues differently, and may vary in terms of their importance in meeting the fatty acid requirements of fish. To address these questions, Atlantic salmon were fed experimental diets containing, as the sole added dietary lipid source, fish oil (positive control), tallow (negative control), or tallow supplemented with EPA, DHA, or both fatty acids to ~50 or 100% of their respective levels in the positive control diet. Following 14 weeks of feeding, the negative control diet yielded optimum growth performance. Though surprising, these results support the notion that Atlantic salmon requirements for n-3 LC-PUFA are quite low. EPA was largely β-oxidized and inefficiently deposited in tissues, and increasing dietary levels were associated with potential negative effects on growth. Conversely, DHA was completely spared from catabolism and very efficiently deposited into flesh. EPA bioconversion to DHA was largely influenced by substrate availability, with the presence of preformed DHA having little inhibitory effect. These results clearly indicate EPA and DHA are metabolized differently by Atlantic salmon, and suggest that the n-3 LC-PUFA dietary requirements of Atlantic salmon may be lower than reported and different, if originating primarily from EPA or DHA.