Recombinant Escherichia coli, expressing the oleate hydratase gene of Stenotrophomonas maltophilia, was permeabilized by sequential treatments with 0.125 M NaCl and 2 mM EDTA. The optimal conditions for the production of 10-hydroxy-12,15(Z,Z)-octadecadienoic acid from α-linolenic acid by permeabilized cells were 35 °C and pH 7.0 with 0.1 % (v/v) Tween 40, 50 g permeabilized cells l−1, and 17.5 g α-linolenic acid l−1. Under these conditions, permeabilized cells produced 14.3 g 10-hydroxy-12,15(Z,Z)-octadecadienoic acid l−1 after 18 h, with a conversion yield of 82 % (g/g) and a volumetric productivity of 0.79 g l−1 h−1. These values were 17 and 168 % higher than those obtained by nonpermeabilized cells, respectively. The concentration, yield, and productivity of 10-hydroxy-12,15(Z,Z)-octadecadienoic acid obtained by permeabilized cells are the highest reported thus far.