A key step in the rational design of new DNA binding agents is to obtain a complete thermodynamic characterization of small molecule-DNA interactions. Ethidium bromide has served as a classic DNA intercalator for more than four decades. This work focuses on delineating the influence(s) of the 3- and 8-amino substituents of ethidium on the energetic contributions and concomitant fluorescent properties upon DNA complex formation. Binding affinities decrease by an order of magnitude upon the removal of either the 3- or 8-amino substituent, with a further order-of-magnitude decrease in the absence of both amino groups. The thermodynamic binding mechanism changes from enthalpy-driven for the parent ethidium to entropy-driven when both amino groups are removed. Upon DNA binding, fluorescence enhancement is observed in the presence of either or both of the amino groups, likely because of more efficient fluorescence quenching through solvent interactions of free amino groups than when buried within the intercalation site. The des-amino ethidium analog exhibits fluorescence quenching upon binding, consistent with less efficient quenching of the chromophore through interactions with solvent than within the intercalation site. Determination of the quantum efficiencies suggests distinct differences in the environments of the 3- and 8-amino substituents within the DNA binding site.