In this paper we report on the enzymatic preparation of D-p-trimethylsilylphenylalanine (D-TMS-Phe). First, DL-5-(p-trimethylsilylphenylmethyl) hydantoin (DL-TMS-Phe-Hyd) was synthesized chemically and subjected to bacterial hydrolysis to obtain N-carbamoyl-D-p-trimethylsilylphenylalanine (C-D-TMS-Phe), but no strains examined showed sufficient hydantoinase activity on this compound. However, Blastobacter sp. A17p-4, which is known to produce N-carbamoyl-D-amino acid amidohydrolase (DCase), was found to be able to hydrolyze C-DL-TMS-Phe prepared chemically from the hydantoin. When C-DL-TMS-Phe was hydrolyzed with cells of Blastobacter sp. A17p-4, its optical purity was low because N-carbamoyl-L-amino acid amidohydrolase (LCase) coexisted in the cells. DCase and LCase in the cell-free extract of Blastobacter sp. A17p-4 could be separated by DEAE-Sephacel column chromatography. The optimum pH for the hydrolysis of C-DL-TMS-Phe by the partially purified DCase was 8.0 and addition of 2.5% N,N-dimethylformamide was effective in raising the substrate concentration without inactivation of DCase. Under the optimized conditions, highly optically pure (98% enantiomeric excess) D-TMS-Phe could be obtained from C-DL-TMS-Phe with partially purified DCase.