To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica. Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 A degrees C, pH 7, 200 rpm with 1.5 g sodium acetate l(-1), 100 g wet cells l(-1), and 25 g LA l(-1). Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l(-1) after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l(-1). Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.