Hybrid tryptophan synthetase alpha and beta polypeptides were produced by genetic recombination between the trpB--trpA regions of Escherichia coli and Salmonella typhimurium contained on compatible, multicopy plasmids. Intragenic recombination was decreased but still evident in recA cells. Genetic exchange occurred at many sites within trpA, but every recombinant gene produced a functional alpha polypeptide despite many amino acid differences from one or the other of the parental polypeptides. The five hybrid tryptophan synthetase alpha subunits examined resembled the parental polypeptides in catalytic function but differed in thermostability. The stability differences suggest that, as amino acid changes occurred in these proteins during the course of evolution, subsequent changes were limited to those that would allow retention of a desired protein conformation.