D-[1-C-13]Glucose is useful for studies on tracing by means of C-13-magnetic resonance imaging (IL.IRI) the fate of a metabolite in the brain, and this technique is expected to become a sophisticated clinical tool for the diagnosis of neurological disorders. For this purpose, a more efficient method of producing inexpensive C-13-labeled D-glucose is necessary, and was investigated. When a mixture of the labeled D-fructose-6-phosphate (F-6-P) and D-glucose-6-phosphate (G-6-P) prepared from D-ribose-5-phosphate and [C-13]formaldehyde is dephosphorylated by potato acid phosphatase, a combination of potato acid phosphatase, phosphoglucose isomerase, and BaCl2 was found to be effective in improving the yield of D-glucose and shortening the reaction time. The maximum yield of labeled D-glucose from the mixture of labeled F-6-P and G-6-P was 84%. Two peaks indicative of labeled D-glucose with C-13 incorporated at the C-l position were confirmed by the NMR spectroscopy.