The monovalent cation (MVC) site of the tryptophan synthase from Salmonella typhimurium plays essential roles in catalysis and in the regulation of substrate channeling. In vitro, MVCs affect the equilibrium distribution of intermediates formed in the reaction of l-Ser with the alpha(2)beta(2) complex; the MVC-free, Cs(+)-bound, and NH(4)(+)-bound enzymes stabilize the alpha-aminoacrylate species, E(A-A), while Na(+) binding stabilizes the l-Ser external aldimine species, E(Aex(1)). Two probes of beta-site reactivity and conformation were used herein, the reactive indole analogue, indoline, and the l-Trp analogue, l-His. MVC-bound E(A-A) reacts rapidly with indoline to give the indoline quinonoid species, E(Q)(indoline), which slowly converts to dihydroiso-l-tryptophan. MVC-free E(A-A) gives very little E(Q)(indoline), and turnover is strongly impaired; the fraction of E(Q)(indoline) formed is <3.5% of that given by the Na(+)-bound form. The reaction of l-Ser with the MVC-free internal aldimine species, E(Ain), initially gives small amounts of an active E(A-A) which converts to an inactive species on a slower, conformational, time scale. This inactivation is abolished by the binding of MVCs. The inactive E(A-A) appears to have a closed beta-subunit conformation with an altered substrate binding site that is different from the known conformations of tryptophan synthase. Reaction of l-His with E(Ain) gives an equilibrating mixture of external aldimine and quinonoid species, E(Aex)(his) and E(Q)(his). The MVC-free and Na(+) forms of the enzyme gave trace amounts of E(Q)(his) ( approximately 1% of the beta-sites). The Cs(+) and NH(4)(+) forms gave approximately 17 and approximately 14%, respectively. The reactivity of MVC-free E(Ain) was restored by the binding of an alpha-site ligand. These studies show MVCs and alpha-site ligands act synergistically to modulate the switching of the beta-subunit from the open to the closed conformation, and this switching is crucial to the regulation of beta-site catalytic activity. Comparison of the structures of Na(+) and Cs(+) forms of the enzyme shows Cs(+) favors complexes with open indole binding sites poised for the conformational transition to the closed state, whereas the Na(+) form does not. The beta-subunits of Cs(+) complexes exhibit preformed indole subsites; the indole subsites of the open Na(+) complexes are collapsed, distorted, and too small to accommodate indole.