An efficient procedure for the production of l-[4- 1 3 C]aspartic acid (4- 1 3 C-Asp) was investigated. In this procedure, phosphoenolpyruvate carboxylase originating from the methanol-assimilating microbe, Methylobacterium extorquens JCM 2805, was used for the production of labeled oxaloacetic acid from phosphoenol-pyruvate (PEP) and NaH 1 3 CO 3 ; the oxaloacetic acid was then converted to 4- 1 3 C-Asp with glutamic-oxaloacetic transaminase. In this reaction, with starting concentrations of 10 mM PEP, 10 mM NaH 1 3 CO 3 and 15 mM l-glutamic acid (Glu), the yield was 70%. 4- 1 3 C-Asp and Glu in the final reaction mixture were separated by displacement chromatography. The yield of this process was 84%. The overall yield was 59%. The incorporation of 1 3 C at the C-4 position of 4- 1 3 C-Asp was confirmed by NMR spectroscopy.