This study describes a methodology with potential application in the estimation of essential fatty acid (EFA) requirements of fish larvae. Senegalese sole (Solea senegalensis) larvae were fed, from 16 days after hatching (DAH), on Artemia enriched with different oils, inducing graded dietary concentrations of DHA: (1) soyabean oil, containing no measurable amounts of DHA (NDHA); (2) fish oil, inducing a medium DHA level (MDHA, 3 g DHA/100 g fatty acids); and (3) a mixture of Easy DHA Selco and Microfeed, resulting in high DHA content (HDHA, 8 g/100 g). At 28 DAH a metabolic trial was conducted where larvae were tube fed [1-14 C]DHA, in order to determine its absorption, retention in the gut and body tissues, as well as its oxidation. At 23 DAH the HDHA treatment induced a significantly higher larval growth, while at 32 DAH significant differences were only found between the NDHA and HDHA treatments. The absorption of tube-fed [1-14 C]DHA was extremely high (94–95 %) and independent of feeding regime. However, in larvae fed NDHA Artemia, a significantly higher amount of label was retained in the gut compartment and a concurrently lower retention was measured in the body. A significantly higher proportion of the absorbed DHA label was oxidized in larvae fed HDHA, compared to NDHA. Based on these results, we suggest that increasing dietary supply of DHA above the larval requirement level results in its increased oxidation for energy purposes and we propose potential applications of the tube feeding methodology using radiolabelled EFA in conjunction with dose–response studies.