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 sequences of DNA, as expected, while results with sequences containing GC or mixed AT and GC sequences support an intercalation mode for these compounds. The weak induced CD signals for the compounds in complex with poly d(G-C)(2), for example, are characteristic of intercalation and the electric linear dichroism spectra demonstrate clearly that the compounds have their transition dipoles oriented in the same plane as the DNA base pairs, exactly as predicted for intercalation binding. Chemical shift changes of the aromatic proton signals of the diphenylfuran ring system are all upfield by approximately 0.5 ppm or greater on complex formation with GC sequences, also as predicted for intercalation. The compounds have NOE contacts to DNA protons in both the major and the minor grooves, and this is only possible if the compounds extend through the DNA double helix, as expected for an intercalation binding mode. Some of the discrepancy in the literature may have arisen due to confusion caused by the mixed minor-groove and intercalation complexes of the diphenylfurans in heterogeneous sequence DNA.