d-Branched-chain amino acids (d-BCAAs) such as d-leucine, d-isoleucine, and d-valine are known to be peptide antibiotic intermediates and to exhibit a variety of bioactivities. Consequently, much effort is going into achieving simple stereospecific synthesis of d-BCAAs, especially analogs labeled with stable isotopes. Up to now, however, no effective method has been reported. Here, we report the establishment of an efficient system for enantioselective synthesis of d-BCAAs and production of d-BCAAs labeled with stable isotopes. This system is based on two thermostable enzymes: d-amino acid dehydrogenase, catalyzing NADPH-dependent enantioselective amination of 2-oxo acids to produce the corresponding d-amino acids, and glucose dehydrogenase, catalyzing NADPH regeneration from NADP(+) and d-glucose. After incubation with the enzymes for 2 h at 65A degrees C and pH 10.5, 2-oxo-4-methylvaleric acid was converted to d-leucine with an excellent yield (> 99 %) and optical purity (> 99 %). Using this system, we produced five different d-BCAAs labeled with stable isotopes: d-[1-C-13,N-15]leucine, d-[1-C-13]leucine, d-[N-15]leucine, d-[N-15]isoleucine, and d-[N-15]valine. The structure of each labeled d-amino acid was confirmed using time-of-flight mass spectrometry and nuclear magnetic resonance analysis. These analyses confirmed that the developed system was highly useful for production of d-BCAAs labeled with stable isotopes, making this the first reported enzymatic production of d-BCAAs labeled with stable isotopes. Our findings facilitate tracer studies investigating d-BCAAs and their derivatives.