Five experimental diets with constant total C(18) PUFA and varying 18:3n-3/18:2n-6 ratios were fed to rainbow trout over an entire production cycle. The whole-body fatty acid balance method demonstrated a clear trend of progressively reduced fatty acid bioconversion activity along the n-3 and n-6 pathways, up to the production of 20:5n-3 and 20:4n-6, respectively. This suggests that the pathway exhibits a "funnel like" progression of activity rather than the existence of a single rate limiting step. The production of 22:5n-3 and 22:6n-3 was more active than that of 20:5n-3. However, despite this trend in reduced apparent in vivo net enzyme activity, the efficiency of the various bioconversion steps (measured as % of bioconverted substrate) confirmed an opposing trend. A 3.2-fold higher Δ-6 desaturase affinity towards 18:3n-3 over 18:2n-6 and an 8-fold greater Δ-5 desaturase affinity towards 20:4n-3 over 20:3n-6 were recorded. The main results of the study were that (1) rainbow trout are quite efficient at bioconverting 18:3n-3 to 22:6n-3, and (2) the LC-PUFA biosynthetic pathway is substrate limited. Fillet n-3 LC-PUFA concentrations increased with the increasing dietary supply of 18:3n-3. Despite an almost identical dietary supply of n-3 LC-PUFA, originating from the fish meal fraction of the diets, the fillets of trout fed the diet richest in 18:3n-3 were 2-fold higher in n-3 LC-PUFA than fish fed low 18:3n-3 diets. Nevertheless, fillets of trout fed a fish oil control diet contained more than double the amount of n-3 LC-PUFA compared to fish fed the diets richest in 18:3n-3.