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Binding of tryptophan, tryptamine and ATP to tryptophanyl-tRNA synthetase was studied by equilibrium dialysis experiments. There arc two binding sites per mole of enzyme both for tryptophan and for tryptamine. Ks for tryptophan is 0.95 μM and for tryptamine is 1.8 μM. In the case of ATP no binding could he measured over the range of concentrations examined. The Scatchard plots of tryptophan and tryptamine binding do not shown any cooperativity between the subunits. Dissociation of the dimeric enzyme was studied at very low protein concentration. Upon dilution of the enzyme both the [32P]PPi-ATP isotope exchange activity and the tRNA charging activity are lost simultaneously. Kinetic evidence demonstrates that the inactivation is primarily due to the dissociation of the active direct into inactive monomers. The dissociation is strongly promoted by alkaline pH and is inhibited by the presence of tryptophan or tryptophanyladenylate but not by that of tRNA. The dissociation constant of the dimer-monomer equilibrium at pH 8.5 is 15 nM at 25 °C. The dissociation form is more susceptible to denaturation than the dimeric species. The results reported in the paper suggest that even if there are not binding interactions between the subunits, the active conformation of the enzyme depends on their associated state. [ABSTRACT FROM AUTHOR]