Using circular dichroism, differential scanning calorimetry and susceptibility to DNAse I cleavage assays, we show that the interaction of berenil, a minor-groove binding drug, with poly(dA-dT).poly(dA-dT) and poly(dA).poly(dT) involves important changes in the polynucleotide conformation. The effect of berenil on poly(dA-dT).poly(dA-dT) comprises a clear alteration in CD spectra even at drug/DNA ratios smaller than the stoichiometric value. Berenil recognizes and binds to the alternating-B conformation of DNA changing it to a new conformation which appears to show some of the peculiarities of poly(dA).poly(dT), possibly through a modification in the helical parameters at the TpA and ApT steps. Such alteration is accompanied by a small calorimetric enthalpy change. Moreover, the calorimetric enthalpy does not change significantly whatever the input ratio of drug to poly(dA-dT).poly(dA-dT), indicating that berenil binding does not substantially alters the enthalpy of transition. In addition to increasing the melting temperature of the polynucleotide, berenil reduces the cooperativity of the poly(dA-dT).poly(dA-dT) transition slightly more than either distamycin or netropsin.