The high mortalities observed during Octopus vulgaris paralarvae culture have been associated with a nutritional imbalance, with long-chain polyunsaturated fatty acids (LC-PUFA) appearing to have a critical role. In order to determine the in vivo capability of O. vulgaris hatchlings to incorporate and metabolise unsaturated fatty acids (FA), hatchlings were incubated in flat-bottom 6-well tissue culture plates at a density of 90 hatchlings/well in 10 mL of seawater (36‰). Incubations were performed with gentle stirring at 21 °C for 6 h with 0.2 μCi (0.3 μM) of [1−14C]-labelled FA including 18:1n−9, 18:2n−6, 18:3n−3, 20:4n−6 (ARA), 20:5n−3 (EPA) or 22:6n−3 (DHA), which were added directly to the seawater as their potassium salts bound to bovine serum al- bumin (BSA). A control treatment without [1−14C]FA was also assessed. O. vulgaris hatchlings not only possessed the ability to incorporate FA bound to BSA, but also to esterify them into phospholipid, with marked specificity. [1−14C]DHA and [1−14C]C18 FA substrates were mainly esterified into phosphatidylcholine, while [1−14C]ARA and [1−14C]EPA were esterified into phosphatidylethanolamine. The majority of radioactivity from [1−14C]FA incorporated into hatchling total lipid was recovered as unmodified FA with elongation being the only metabo- lism detected. Of the FA investigated, [1−14C]ARA was the most efficiently incorporated into hatchling lipids, but it was also the least modified FA. The fact that no desaturation activity was recorded towards the FA tested in this experiment may indicate that the nutritional requirements of O. vulgaris hatchlings in terms of FA are highly specific and LC-PUFA must be considered essential dietary nutrients.