Tryptophan synthase (EC 4.2. 1.20) from bacteria, yeasts, molds, and plants catalyzes the final two reactions in the biosynthesis of L-tryptophan. This enzyme has been the subject of many important genetic and biochemical studies and has been frequently reviewed (1-5). I emphasize here the important progress made since my previous review in this series in 1979 (2). I describe the three-dimensional structure of the tryptophan synthase azẞ2 complex from Salmonella typhimurium (6, 7) and correlate this new structural information with previous biochemical and genetic studies. I describe how site-directed mutagenesis is being used to explore the relationship between enzyme structure and enzyme mechanism. The early history of the studies of tryptophan synthase from Neurospora crassa and from Escherichia coli has been vividly recounted by Yanofsky (8, 9). Studies of mutants that require tryptophan for growth led to the discovery that tryptophan synthase from E. coli is a multifunctional, multicomponent enzyme (9, 10). Enzyme fractionation demonstrated that the enzyme from E. coli is an α2ß2 complex composed of two nonidentical dissociable subunits, now called the a and B subunits (11). Whereas the isolated a subunit is a monomer, the B subunit is usually a dimer and is often called the ẞ2 subunit. In this chapter I use the term B subunit to refer to each ẞ polypeptide chain and to the active ẞ2 dimer. Figure 1 summarizes the subunit structure of bacterial tryptophan synthase and the reactions involved in the synthesis of L-tryptophan. Although the separate a and B subunits have low activities in the a and ẞ reactions, respectively, the a2ß2 complex has much higher activities in these reactions. The a2ß2 complex also has a higher affinity for substrates than the separated a and ẞ subunits. The physiologically important reaction catalyzed by the α2ß2 complex, termed here the aß reaction, is the sum of the a and ẞ reactions. In the overall aẞ reaction, indole produced in the active site of the a subunit becomes a substrate for the B subunit, where it is converted to L-tryptophan by a pyridoxal phosphate-dependent B-replacement reaction with L-serine. Although early experiments showed that indole does not appear as a free intermediate in the aß reaction (12–14), these results could not distinguish whether the sites at which the a and ẞ reactions were catalyzed were juxtaposed or connected by a channel. The presence of a channel or tunnel has recently been established by the crystallographic studies (6) to be described in Section II. B.