Dihydroxyacetone synthase (EC 2.2.1.3), which is a key enzyme of the C-1-compound-assimilating pathway in yeasts, catalyzes transketolation between formaldehyde and hydroxypyruvate, leading to the formation of dihydroxyacetone and CO2. When [C-13]formaldehyde was used as a substrate with dihydroxyacetone synthase from Candida boidinii 2201, C-13 was confirmed to be incorporated to the C-1 and C-3 positions of dihydroxyacetone, and the C-13 content of each carbon (atoms/100 atoms) was estimated to be 50%. [C-13]Methanol was also useful for the enrichment of dihydroxyacetone with C-13 : when alcohol oxidase from a methylotrophic yeast was added for the conversion of methanol to formaldehyde. A fed-batch reaction with periodic addition of the substrates was required for the accumulation of C-13-labelled dihydroxyacetone at a higher concentration, because the enzyme system was relatively susceptible to the C donor, formaldehyde or methanol. The optimum conditions for the production gave 160 mM (14.4 mg/ml) dihydroxyacetone for 180 min; the molar yield relative to methanol added was 80%. Dihydroxyacetone kinase (EC 2.7.1.29) from methanol-grown Hansenula polymorpha CBS 4732 was a suitable enzyme for the phosphorylation of dihydroxyacetone. The phosphorylation system, comprising of dihydroxyacetone kinase, adenylate kinase, and ATP, could be coupled with the system for dihydroxyacetone production. A fed-batch reaction afforded 185 mM [1 : 3-C-13]dihydroxyacetone phosphate from [C-13]methanol; the molar yield of the ester relative to methanol added was 92.5%.