C-13-labelled L-serine was efficiently produced from [C-13] formaldehyde and glycine by using partially purified L-serine hydroxymethyltransferase from Methylobacterium extorquens JCM 2804, tetrahydrofolate and a tetrahydrofolate regeneration system. The tetrahydrofolate regeneration system allowed efficient L-serine production and direct use of dihydrofolate easily prepared from folic acid. A tetrahydrofolate concentration of more than 0.020% was necessary for efficient production. Eighty seven % of the amount of [C-13] formaldehyde consumed, i.e., the difference between the total amount of formaldehyde fed and the amount of formaldehyde remaining in the reaction mixture, was used for the incorporation into L-[C-13] serine. Seventy two mg of added glycine was converted to L-serine when 100 mg of glycine was contained in 11.2 ml of the final reaction mixture. The L-serine was confirmed to be L-[3-C-13]-serine by negative fast atom bombardment mass spectrometry (FAB-MS) and C-13-nuclear magnetic resonance spectrometry (NMR) analysis.