Phenylalanine dehydrogenase of Thermoactinomyces intermedius acts preferentially on L-phenylalanine and L-tyrosine, whereas leucine dehydrogenase of Bacillus stearothermophilus acts almost exclusively on L-leucine and some other branched-chain L-amino acids. The two enzymes share a sequence similarity (47%). Aiming at elucidation of the mechanism of substrate recognition by the two amino acid dehydrogenases, we have genetically constructed a chimeric enzyme consisting of an N-terminal domain of phenylalanine dehydrogenase containing the substrate-binding region and a C-terminal domain of leucine dehydrogenase containing the NAD(+)-binding region. The chimeric enzyme purified to homogeneity acted on phenylalanine with a specific activity of 6% of that of the parental phenylalanine dehydrogenase and showed a broad substrate specificity in the oxidative deamination, like phenylalanine dehydrogenase. However, it acted much more effectively than phenylalanine dehydrogenase on isoleucine and valine. Its Km values for L-phenylalanine and L-leucine were similar to those of phenylalanine dehydrogenase. The substrate specificity of the chimeric enzyme in the reductive amination was an admixture of those of the two parent enzymes. These results suggest that the two domains of phenylalanine dehydrogenase and leucine dehydrogenase probably can fold independently. Accordingly, their chimera forms a new active enzyme which consists of their N- and C-terminal domains containing the substrate- and coenzyme-binding regions, respectively. However, the two domains of chimeric enzyme interact and communicate with each other to form a new active site and consistently show the new substrate specificity.