It has been known that transfer RNA assumes a functional conformation in the presence of certain metal ions, most commonly magnesium ions. Several Mg²⁺-binding sites of high affinity have been revealed by X-ray crystallographic analyses of yeast tRNA^Phe. On the other hand, ATP, a naturally occurring high-energy compound and which is one of the substrates in aminoacylation of tRNA, is known to have ability to chelate Mg²⁺. We therefore examined the possibility that the conformation of tRNA is regulated by the quantitative balance between Mg²⁺ and ATP, and consequently biological activity of tRNA is modulated as well. Structural transition of tRNA was monitored by measuring the melting profiles of yeast tRNA^Tyr in the presence of Mg²⁺ and/or ATP at various [ATP]/[Mg²⁺] ratios. Tyrosine-accepting activity of yeast tRNA^Tyr was also measured under the similar solution conditions. These results clearly indicated that conformation of tRNA and its aminoacylation capacity can be easily modulated by changing the relative concentration ratio of Mg²⁺ and ATP.