Inhibitors of beta-oxidation on the synthesis of glycolipid biosurfactant, mannosylerythritol lipid (MEL), were used to clarify the fatty acid metabolism of MEL in Candida antarctica. 2-Bromooclanoic acid drastically inhibited the lipid synthesis under growing- and resting-cell conditions; moreover, the degree of the inhibition increased along with increases in both the inhibitor concentration and the chain-length of the fatty acid substrate used. These results clearly provide additional support for the essential contribution of the mammalian type of ＇chain-shortening pathway＇ (partial beta-oxidation) to the biosynthesis of the extracellular glycolipids.