The binding of the various substrates to Escherichia coli glutamyl-tRNA synthetase has been investigated by using as experimental approaches the binding study under equilibrium conditions and the substrate-induced protection of the enzyme against its thermal inactivation. The results show that ATP and tRNAGlu bind to the free enzyme, whereas glutamate binds only to an enzyme form to which glutamate-accepting tRNAGlu is associated. By use of modified E. coli tRNAsGlu and heterologous tRNAsGlu, a correlation could be established between the ability of tRNAGlu to be aminoacylated by glutamyl-tRNA synthetase and its abilities to promote the [32P]PPi-ATP isotope exchange and the binding of glutamate to the synthetase. These results give a possible explanation for the inability of blutamyl-tRNA synthetase to catalyze the isotope exchange in the absence of amino acid accepting tRNAGlu and for the failure to detect an enzyme-adenylate complex for this synthetase by using the usual approaches. One binding site was detected for each substrate. The specificity of the interaction of the various substrates has been further investigated. Concerning ATP, inhibition studies of the aminoacylation reaction by various analogues showed the existence of a synergistic effect between the adenine and the ribose residues for the interaction of adenosine. The primary recognition of ATP involves the N-1 and the 6-amino group of adenine as well as the 2'-OH group of ribose. This first interaction is then strengthened by the phosphate groups- Inhibition studies by various analogues of glutamate showed a strong decrease in the affinity of this substrate for the synthetase after substitution of the alpha- or gamma-carboxyl groups. The enzyme exhibits a marked tendency to complex tRNAs of other specificities even in the presence of tRNAGlu. MgCl2 and spermidine favor the specific interactions. The influence of monovalent ions and of pH on the interaction between glutamyl-tRNA synthetase and tRNAGlu is similar to those reported for other synthetases not requiring their cognate tRNA to bind the amino acid. Finally, contrary to that reported for other monomeric synthetases, no dimerization of glutamyl-tRNA synthetase occurs during the catalytic process.