A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable beta-cyano-L-alanine synthase. The enzyme catalyzes the synthesis of beta-cyano-L-alanine from O-acetyl-L-serine and cyanide ions. The purified enzyme has a molecular mass of approximately 70 kDa and consists of two identical subunits. It was stable in the pH range of 6.0 to 10.0 and up to 70 degrees C. The enzyme also catalyzes the synthesis of various beta-substituted-L-alanine derivatives from O-acetyl-L-serine and nucleophilic reagents. The gene encoding the beta-cyano-L-alanine synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the beta-cyano-L-alanine synthase of the bacterium is O-acetyl-L-serine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli JM109. The transformed E. coli cells overexpressed beta-cyano-L-alanine synthase. Heat stable beta-cyano-L-alanine synthase can be applied to the synthesis of [4-11C]l-2,4-diaminobutyric acid as a tracer for positron emission tomography.