Synthetic alternating GC-rich DNA polymers can adopt Hoogsteen base-paired structures (H^L-form) under the influence of low pH and temperature. The interaction of aristololactam-β-D-glucoside (ADG), a natural glucoside derivative of aristolochia group of alkaloids, with protonation-induced structures (H^L-form) of poly(dG-dC)·poly(dG-dC) and poly(dG-m⁵dC)·poly(dG-m⁵dC) has been studied using different biophysical techniques. The binding of ADG to protonated DNA is characterized by typical hypochromism and bathochromism of the absorption spectrum of the alkaloid, quenching of steady state fluorescence intensity, decrease in quantum yield, increase in fluorescence polarization anisotropy values, increase in thermal transition temperature of polynucleotides following alkaloid binding and perturbation of circular dichroic spectrum of polynucleotides as a result of its interaction with the alkaloid. Scatchard analysis of the data indicates that ADG binds to protonated structures in a nonlinear noncooperative manner. The binding parameters determined from spectrophotometric titration data employing excluded site model indicate that protonated poly(dG-m⁵dC)·poly(dG-m⁵dC) is more favorable for ADG binding than the corresponding nonmethylated analog. The binding of ADG to protonated structures renders a higher degree of stabilization against thermal denaturation compared to respective B-form-ADG interactions and induces a conformational switch to a bound altered form which is different from its interaction with B- and Z-form DNA structures. Thermodynamic parameters (ΔG°, ΔH° and ΔS°) obtained by van?t Hoff analysis of the data indicate that the binding of alkaloid to protonated structures is an exothermic process and the binding free energy arises primarily from a negative enthalpy change. Moreover, the binding leads to an increase in the contour length of protonated DNAs. These results suggest that ADG possibly binds to protonated DNAs by the mechanism of intercalation.