To investigate the effects of ATP, Mg2+, and a mixture of the two on the formation of large DNA aggregates induced by spermidine, we constructed novel phase diagrams of the spermidine/DNA/Tris-buffer system in the presence of these compounds. These diagrams reveal not only the difference in the inhibition of the aggregate formation between ATP and Mg2+ but also a type of buffering effect whereby the complex formation of ATP and Mg2+ diminishes their individual inhibitory actions. Furthermore, the equilibrium composition of ATP, spermidine, Mg2+, and their complexes in the liquid phase was estimated using the equilibrium constants for the complex formations and their mass balances. From this solution equilibrium analysis, the threshold condition for DNA aggregation is suggested. The phase diagrams and equilibrium analysis presented here are useful to predict the threshold of aggregation in a system similar to in vitro transcription. Finally, we recommend that region b in this paper, where the variation in the concentration of either ATP or Mg2+ had little effect on the DNA aggregation, should be used to establish the initial conditions of in vitro RNA synthesis.