The aminoacylation of tRNA catalysed by valyl-tRNA synthetase (EC 184.108.40.206) and isoleucyl-tRNA synthetase (EC 220.127.116.11) fromMycobacterium smegmatis is dependent on the presence of divalent metal ions. Polyamines alone, in the absence of metal ions, do not bring about aminoacylation. In the presence of suboptimal concentrations of Mg2+, polyamines significantly stimulate the reaction. Of the cations tested, only Mn2+, Co2+ and Ca2+ can partially substitute for Mg2+ in aminoacylation, and spermine stimulates aminoacylation in the presence of these cations also. At neutral pH, spermine deacylates nonenzymatically aminoacyl tRNA. AMP and pyrophosphate-dependent enzymatic deacylation of aminoacyl-tRNA (reverse reaction) is also stimulated by spermine. The inhibitory effect of high concentration of KC1 on aminoacylation is counteracted, by spermine. The low level of activity between pH 8.5–9.0 at 1.2 mM Mg2+ is restored to normal level on the addition of spermine. The inhibitory effect of high pH on aminoacylation in the presence of low concentration of Mg2+ is also prevntedvby spemine.