The effects of spermine have been studied on the aminoacylation reaction catalyzed by rat liver threonyl-tRNA synthetase. Spermine can not replace Mg2+ in this reaction. However, a stimulatory and synergistic effect was observed on the threonyl-tRNA formation, in the presence of spermine and suboptimal concentration of Mg2+. Other divalent cations like Ba2+, Ca2+, Mn2+ and Co2+ can substitute Mg2+ in the threonyl-tRNA formation, but in all these cases spermine had no significant effect. Spermine prevented the inhibitory effects caused by excess of ATP or tRNA on the aminoacylation reaction. Association constants were determined by equilibrium dialysis for the tRNA-spermine complex (Ka = 3.7 x 10(3) M-1) and by differential spectrophotometry for the ATP-spermine complex (Ka = 7.8 x 10(3) M-1). No enzyme-spermine complex could be detected by equilibrium dialysis. Some roles have been ascribed for the polyamine spermine in the stimulation of the threonyl-tRNA formation. ATP-spermine and tRNA-spermine can not function as substrates for the threonyl-tRNA synthetase, since Mg2+ is indispensable. The stimulatory effect by spermine is important considering the physiological concentration of Mg2+ in the tissues. Probably in vivo spermine would have a relevant role lowering the real Mg2+ concentration required in the aminoacylation reaction.7433-43