The interaction of thionine with triple, double, and single RNA helices has been fully characterized by thermodynamic and kinetic methods. The nature of the interaction of thionine with the synthetic polynucleotides poly(rU), poly(rA)center dot poly(rU), and poly(rA)center dot 2poly(rU) has been studied at pH = 7.0 and 25 degrees C by UV absorbance, fluorescence, circular dichroism spectroscopy, viscometry, differential scanning calorimetry, and T-jump kinetic measurements. The results show that at I = 0.1 M thionine binds to a single poly(rU) strand, destabilizes the poly(rA)center dot 2poly(rU) triplex by external binding, and intercalates into poly(rA)center dot poly(rU) with similar affinity to the thionine/DNA intercalated complex (Paul, P.; Kumar, G. S. J. Fluoresc. 2012, 22, 71-80). On the other hand, the differential scanning calorimetry measurements performed with thionine display a point in which the heat capacity remains unaltered, revealing the equilibrium of isothermal denaturation: thionine/poly(rA)center dot 2poly(rU) + thionine (sic) thionine/poly(rA)center dot poly(rU) + thionine/poly(rU), an outcome supported by the other techniques used. The denaturation equilibrium constant, K-D (25 degrees C) = 522 M-1, was evaluated from the affinity with the single, duplex, and triplex RNA.