The transition of Sepia officinalis culture to industrial large scale has been hampered due to bottlenecks related to the limited knowledge on nutritional physiology of the species. Determination of the endogenous ability of S. officinalis hatchlings to metabolise unsaturated fatty acids (FA) may provide new insight on the capability of hatchlings to biosynthesise different FA, as well as lipid classes containing essential fatty acids (EFA). In the present study, cuttlefish hatchlings were incubated with [1-14C]FA including C18 FA (18:1n-9, 18:2n-6, 18:3n-3) and long-chain polyunsaturated fatty acids (LC-PUFA) (20:4n-6 (ARA), 20:5n-3 (EPA) or 22:6n-3 (DHA)), which were added individually as potassium salts bound to bovine serum albumin. The majority of radioactivity incorporated was recovered esterified into polar lipids (PL). A pattern was detected, where [1-14C]DHA, [1-14C]C18 FA and their metabolic products were preferentially esterified into phosphatidylcholine, whereas [1- 14C]ARA and [1-14C]EPA were mainly esterified into phosphatidylethanolamine. [1-14C]C18 FA were the most transformed FA with several metabolites produced by elongation and possible desaturation being obtained. Of the radioactivity incorporated into hatchling total lipid (TL) from supplemented [1- 14C]LC-PUFA only one elongation product was recovered from the three substrates, except for [1- 14C]ARA, where an unidentified product was also detected. The present in vivo results indicated that S. officinalis hatchlings may have capability for the first steps in the biosynthesis of ARA and EPA from 18:2n-6 and 18:3n-3, respectively, including the existence of a desaturase potentially involved. Nonetheless, considering the low desaturation rates detected, this process may not be sufficient to cover EFA demands during development of this species. Therefore, dietary ARA and EPA, as well as DHA, should be supplied during the hatchling stage of Sepia. While designing an inert diet, which ensures normal growth and development of this species during the hatchling stage, the C18 FA and LCPUFA levels and ratios should be considered, since the esterification pattern detected in the present study suggested competition between these FA for esterification into specific lipid classes. Moreover, considering the observed esterification pattern of LC-PUFA into different lipid classes, it is likely that the DHA/EPA/ARA ratio, rather than DHA/EPA or EPA/ARA ratios, would be of great importance for S. officinalis hatchling development.