Tryptophan synthase (TrpS) is a pyridoxal phosphate-containing bifunctional enzyme that catalyzes the last two steps in the biosynthesis of L-tryptophan. Indole, an intermediate generated at the active site of the alpha-subunit is channeled via a 25 A long tunnel to the beta-active site where it reacts with an aminoacrylate intermediate derived from L-serine. The two reactions are kept in phase by allosteric interactions between the two subunits. The recent development of novel alpha-site ligands and alpha-reaction transition state analogs combined with kinetic and crystal structure analysis of Salmonella typhimurium tryptophan synthase has provided new insights into the allosteric regulation of substrate channeling, the reaction mechanisms of the alpha and beta active sites, and the influence of structural dynamics.