An efficient procedure for the production of L-[4-C-13]aspartic acid (4-C-13-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 phosphoenolpyruvate (PEP) and (NaHCO3)-C-13; the oxaloacetic acid was then converted to 4-C-13-Asp with glutamic-oxaloacetic transaminase. In this reaction, with starting concentrations of 10 mM PEP, 10 mM (NaHCO3)-C-13 and 15 mM L-glutamic acid (Glu), the yield was 70%. 4-C-13-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 C-13 at the C-4 position of 4-C-13-Asp was confirmed by NMR spectroscopy.