Amidine derivatives of the 2,5-diphenylfuran aromatic system have activity against a variety of microorganisms. The compounds bind strongly to continuous sequences of AT base pairs in DNA, and there is general agreement that the compounds complex in the minor groove in AT sequences. Some of the derivatives also bind strongly in GC rich and mixed sequences of DNA, and both major-groove and intercalation binding modes have been suggested for this binding mode on the basis of different experimental observations. To obtain definitive information on the DNA binding modes of these types of compounds, we have synthesized additional derivatives, which were designed to provide improved distinction between major-groove and intercalation binding modes, and have extended the experimental analysis to include electric linear dichroism, high-resolution NMR, and absorption, fluorescence, and CD spectroscopy results. All of the spectral results as well as kinetics studies results support a minor-groove binding mode in AT ...