The physiological concentration of free magnesium in Escherichia coli cells is about 1 mM, and there is almost no chloride in the cell. When the aminoacylation of tRNA by tyrosyl-tRNA synthetase was assayed at 1 mM free Mg 2 + , chloride (and sulphate) ions inhibited the reaction but acetate at the same concentration (<200 mM) was not inhibitory. When the magnesium concentration was increased to 10 mM there was almost no chloride inhibition any more. Chloride strengthened the PP i inhibition, the K a p p i (PP i ) values at 1 mM free Mg 2 + were 140, 120, and 56 μM at 0, 50 and 150 mM KCl, respectively. Chloride weakened the AMP inhibition, the corresponding values for K a p p i (AMP) were 0.35, 0.5, and 0.9 mM. The value of K a p p m (tRNA T y r ) was clearly increased by chloride, being 22, 37, 93, and 240 nM at 0, 50, 100, and 150 mM KCl, respectively. Best-fit analyses of the PP i inhibition, AMP inhibition and K a p p m (tRNA) assays were accomplished using total rate equations. The analysis showed that the only kinetic events which are obligatory to explain the chloride effects are a weakened binding of Mg 2 + to the tRNA before the transfer reaction and a weakened binding of Mg 2 + to the Tyr-tRNA.enzyme complex after the transfer reaction. The dissociation constants for the former were 0.11, 0.3, and 2.8 mM and for the latter 0.6, 2.5, and 13 mM at 0, 50 and 150 mM KCl, respectively. Mg 2 + is required for the reactive conformation of tRNA in the transfer reaction but chloride weakens its formation. After the transfer reaction the dissociation of Mg 2 + from the aa-tRNA.enzyme complex enhances the dissociation of the aa-tRNA from the enzyme. The kinetics and the chloride effect were similar in the tyrosyl-tRNA synthetases from both Bacillus stearothermophilus and E. coli.