Optically active p-trimethylsilylphenylalanine (TMS-Phe) was prepared by enantioselective hydrolysis of N-carbamoyl-DL-p-trimethylsilylphenylalanine (C-DL-TMS-Phe) with the cell-free extract of Blastobacter sp. A17p-4, which is known to produce N-carbamoyl-D-amino acid amidohydrolase (DCase). Although this bacterium also produced N-carbamoyl-L-amino acid amidohydrolase (LCase), heat treatment of the cell-free extract for 40 min at 50 degrees C and pH 7.0 was found to be effective in completely inactivating the LCase without loss of DCase activity, which provided a far simpler and more convenient method of preparing optically pure D-TMS-Phe (99% enantiomeric excess, ee). Furthermore, optically pure L-TMS-Phe (99% ee) could be obtained by LCase-catalyzed hydrolysis of the residual substrate with the non-treated cell-free extract as the enzyme source. The optimum pH for the hydrolysis of C-L-TMS-Phe was 7.0, and addition of 2 mM Mn2+ and 5% N,N-dimethylformamide were effective in accelerating the activity of LCase and raising the substrate concentration, respectively.