The pathway for the biosynthesis of docosahexaenoic acid (22:6n-3) fromlinolenic acid (18:3n-3) was investigated in rainbow trout (Oncorhynchus my kiss)liver in vitro, using primary cultures of hepatocytes and liver microsomes toinvestigate the products of desaturation and elongation of [l-14C]-18:3n-3 and[l-14C]-20:5n-3. Argentation thin-layer chromatography and radio gaschromatography were employed to analyse the metabolic products of the radiolabelledfatty acid substrates and to determine the rate of conversion of 18:3n-3 to 22:6n-3. The recovery of radioactivity in various polyunsaturated fatty acids (PUFA) of trouthepatocyte lipid, including 20:5n-3 and 22:6n-3, established that juvenile trout werecapable of converting 18:3n-3 to 22:6n-3. To establish the extent to which theformation of 22:6n-3 was enhanced in the absence of dietary PUFA, particularly20:5n-3 and 22:6n-3, and therefore, facilitate the investigation of 22:6n-3biosynthesis, rainbow trout were fed a diet based on olive oil and deficient in (n-3) PUFA. Feeding the diet deficient in (n-3) PUFA efficiently reduced the endogenouslevels of (n-3) PUFA in trout hepatocytes and at the same time markedly increased therate of 22:6n-3 formation from both [l-14C]-18:3n-3 and [l-14C]-20:5n-3. When thedesaturation and elongation of [1-14C]-18:3n-3 and [l-14C]-20:5n-3 were investigatedin microsomes isolated from trout liver, no radioactivity from either substrate wasrecovered in 22:6n-3. High proportions of radioactivity from [l-14C]-20:5n-3 were,however, recovered in 24:6n-3 and 24:5n-3. These radiolabelled C24-PUFA producedby the microsomal incubations were separated by argentation chromatography andused as substrates in incubations with hepatocytes isolated from trout liver. Docosahexaenoic acid (22:6n-3) was generated from both radiolabelled C24-PUFAsubstrates by trout hepatocytes. The results establish that the biosynthesis of 22:6n-3in rainbow trout hepatocytes does not involve A4-desaturation of 22:5n-3 but ratherproceeds via the A6-desaturation of 24:5n-3 with the subsequent chain shortening ofthe 24:6n-3 produced. Cyclopropene fatty acids and curcumin significantly inhibited the desaturation and elongation activity of hepatocytes and liver microsomes fromrainbow trout. The A6 and A5-desaturase and elongase substrate specificities wereinvestigated; it was shown that (n-3) PUFA substrates were always preferred by theenzymes over (n-6) PUFA.