Differential UV spectroscopy was used to study the temperature dependence of the conformational equilibrium in aqueous poly I · poly A · poly I (A2I) solutions containing Na+ (0.1–2 M) and Mg2+ (10−5–0.005 M) ions. Over the whole range of the studied Na+ and Mg2+ concentrations, the heating‐induced destruction of the triple A2I helix is actually the A2I → A + I + I (3 → 1) transition. The rise of the transition temperature with increasing Na+ and Mg2+ contents is well described by Manning's and the “ligand” theories, which makes it possible to estimate the linear charge density on the single‐stranded poly I (ξ = 1.9 ± 0.1) and the Mg2+–A2I binding constant (K = 1 250 M−1 for the zero degree of binding). An analytical expression has been obtained, which correlates the constants of Mg2+ binding to three‐ and single‐stranded polynucleotides (K3 and K1, respectively) and the linear charge density on them. There are only minor distinctions between the K3 values for A2U and A2I because these polynucleotides have similar structures. The difference in the K1 values is also slight as single‐stranded poly U, poly I, and poly A have similar conformations. Dependence of the conformational transition temperatures of two triple helices with changing Mg2+ concentration.