We constructed an efficient system for preparing optically active 3-trimethylsilylalanine (TMS-Ala) by kinetic resolution with acylase I (aminoacylase; N-acylamino-acid amidohydrolase, EC 3.5.1.14). Racemic TMS-Ala was chemically synthesized and acetylated. Enantioselective deacetylation of N-acetyl-DL-TMS-Ala with acylase I from porcine kidney or from Aspergillus melleus was then attempted. Both enzymes could catalyze the deacetylation of N-acetyl-DL-TMS-Ala, and the porcine enzyme was found to have much higher activity than the enzyme from A. melleus. The optimum pH of the porcine-acylase-catalyzed reaction was 7.5, and the addition of 0.5 mM Co2+ accelerated the reaction. Optically pure L-TMS-Ala (> 99% enantiomeric excess, eel was obtained in 72% yield under the optimized conditions. Furthermore, highly optically pure D-TMS-Ala (96% ee) could also be obtained in 76% yield by chemically hydrolyzing the residual substrate.