Palmitoleic acid (POA) has recently gained attention for its health benefits and as a potential resource for industrial feedstock. This study focused on the use of Saccharomyces cerevisiae, which has a high POA content but low lipid content, for POA production. We created an oleaginous S. cerevisiae as a dga1 mutant overexpressing Dga1p lacking the N-terminal 29 amino acids (Dga1a dagger Np). This was performed to further increase POA content in the oleaginous S. cerevisiae through optimization of culture conditions and genetic modifications. We found that high concentrations of methionine (2.0 g/l) increased POA production in a concentration-dependent way, while other amino acids such as cysteine, glycine, and glutamine showed no effect. It was not clear if the effect of methionine was mediated through S-adenosylmethionine, mainly because its addition did not increase POA content as did the addition of methionine. We increased POA content up to 55 % by incubation of the dga1 transformant in a medium containing 2 g/l methionine at lower than normal temperatures ranging from 20 to 25 A degrees C. Cultivation at such temperatures increased dry cell weight, but did not affect the lipid content, thereby increasing total POA production. The effects of methionine and low temperatures (20-25 A degrees C) on POA content were more apparent in the strains overexpressing Dga1a dagger Np than those harboring empty vectors, which was consistent with the observation that POA was enriched in triacylglycerol. Overexpression of Ole1p, the enzyme responsible for POA production, did not increase POA content of the dga1 mutant overexpressing Dga1a dagger Np, but increased that of the wild-type strain overexpressing Dga1a dagger Np. The results suggested that genomic Ole1p in the dga1 mutant was active enough to achieve the optimal POA production under these conditions. Finally, the POA production by the S. cerevisiae transformant was increased 2.5-fold, which demonstrates that oleaginous S. cerevisiae is a potential source of POA.