The present study presents the first "in vivo" evidence of enzymatic activity and nutritional regulation of a Δ4-desaturase-dependent DHA synthesis pathway in the teleost Solea senegalensis. Juvenile fish were fed diets containing 2 lipid levels (8 and 18%, LL and HL) with either 100% fish oil (FO) or 75% of the FO replaced by vegetable oils (VOs). Fatty acyl elongation (Elovl5) and desaturation (Δ4Fad) activities were measured in isolated enterocytes and hepatocytes incubated with radiolabeled α-linolenic acid (ALA; 18:3n-3) and eicosapentaenoic acid (EPA; 20:5n-3). Tissue distributions of elovl5 and Δ4fad transcripts were also determined, and the transcriptional regulation of these genes in liver and intestine was assessed at fasting and postprandially. DHA biosynthesis from EPA occurred in both cell types, although Elovl5 and Δ4Fad activities tended to be higher in hepatocytes. In contrast, no Δ6Fad activity was detected on (14)C-ALA, which was only elongated to 20:3n-3. Enzymatic activities and gene transcription were modulated by dietary lipid level (LL>HL) and fatty acid (FA) composition (VO>FO), more significantly in the liver than in the intestine, which was reflected in tissue FA compositions. Dietary VO induced a significant up-regulation of Δ4fad transcripts in the liver 6h after feeding, whereas in fasting conditions the effect of lipid level possibly prevailed over or interacted with FA composition in regulating the expression of elovl5 and Δ4fad, which were down-regulated in the liver of fish fed the HL diets. Results indicated functionality and biological relevance of the Δ4 LC-PUFA biosynthesis pathway in S. senegalensis.