The fungal strain Mortierella alliacea YN-15 is a promising industrial producer of polyunsaturated fatty acids (PUFAs), in particular arachidonic acid. In order to more efficiently produce PUFAs, the metabolism of an externally supplied plant oil, α-linolenic acid (ALA)-rich linseed triacylglycerol (TAG), was examined, and time-dependent changes in the composition of its lipid and fatty acid metabolites were traced. Addition of linseed TAG to growing cultures resulted in a transient increase in extracellular 1,2-diacylglycerol (DAG), and even more so of 1,3-DAG, in the mycelia. This was followed by a decrease in both DAGs and an increase in TAG. Eicosapentaenoic acid (EPA), a desaturated and elongated product of ALA, accumulated to a greater extent in cellular phospholipids than in neutral lipids. Moreover, the addition of ALA in free fatty acid form to the culture led to the generation of EPA. However, EPA production was not observed upon addition of ALA-rich 1,2- or 1,3-DAG, indicating that fatty acids released from exogenous lipids were used for resynthesis of mycelial TAG. These results suggested that TAG might be hydrolyzed by extracellular lipases, whereas its synthesis might be catalyzed by intracellular enzymes. Appropriate regulation of such enzymes might be an effective strategy to enhance PUFA production under plant oil supplementation.