Tryptophanase, an enzyme with extreme absolute stereospecificity for optically active stereoisomers, catalyzes the synthesis of L-tryptophan from L-serine and indole through a beta-substitution mechanism of the ping-pong type, and has no activity on D-serine. We previously reported that tryptophanase changed its stereospecificity to degrade D-tryptophan in highly concentrated diammonium hydrogen phosphate, (NH4)(2)HPO4 solution. The present study provided the same stereospecific change seen in the D-tryptophan degradation reaction also occurs in tryptophan synthesis from D-serine. Tryptophanase became active to D-serine to synthesize L-tryptophan in the presence of diammonium hydrogen phosphate. This reaction has never been reported before. D-serine seems to undergo beta-replacement via an enzyme-bonded alpha-aminoacylate intermediate to yield L-tryptophan.