Dietary essential fatty acids may affect larval skeletal formation. The aim of this study was to compare effects on growth and osteological development of dietary docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) being incorporated in the phospholipid (diets PL1 and PL3) or in the neutral lipid (diet NL1) fraction of the larval diet for Atlantic cod (Gadus morhua). The diets were labelled according to the estimated percentage of total n-3 fatty acids contained in the dietary neutral lipid (NL1-1.3% of dietary dry matter) or in the phospholipid fraction (PL1-1.1% and PL3-2.3%). Larvae were weaned to the isoenergetic and isolipiclic microdiets from 17 days post hatching (dph). They were co-fed enriched rotifers until 24 dph and received thereafter only the experimental diets until 45 dph (at 12 degrees C). Dry weight on 45 dph was significantly higher in the PL1- and PL3-larvae (mean 2.74 +/- 0.2 mg DW) than in the NL1-larvae (mean 2.17 +/- 0.1 mg DW). Larvae fed the PL3-diet had a significantly higher DHA content than larvae from the other treatments. No differences were found in larval content of EPA and arachidonic acid (ARA). Larvae fed both PL-diets showed a significantly faster ossification of the vertebral column compared to larvae fed the NL1-diet, according to both larval size and age. On 45 dph, PL3-larvae also had a significantly higher number of fin rays than the other groups at comparable larval sizes. Deformities (mainly kyphosis and shortened vertebrae) were observed in 11% (all groups) of the larvae on 45 dph, with no significant differences among the groups. Ossification of the vertebral column in the cod larvae was first observed in the neural arches on the anterior part of the spine (21 dph, >6.9 mm standard length, SL.), followed by the vertebrae (25 dph, >7.9 mm SL.), haemal arches (31 dph, >= 8.4 mm SL.) and parapophysis (35 dph. >8.7 mm SL). Ossification of all vertebral elements was observed in 45-day-old larvae >= 11.4 mm SL. Large variations between larvae in number of ossifying structures between 31 and 35 dph demonstrated that this was the period of most rapid skeletal change (size range 8.2-11.4 mm SL.), and that the ossification process was more susceptible to fatty acid lipid source rather than to quantity of dietary fatty acids. We suggest that an optimal dietary content of n-3 HUFA in feed for cod larvae is higher than in the PL1-diet. (c) 2009 Elsevier B.V. All rights reserved.