Intercalation mechanisms into double-stranded calf thymus DNA have been probed with 38 different ligands, largely based on naphthalene or quinoline systems. It is shown how NMR shift and line width changes of the ligand signals can be used to unequivocally differentiate intercalation from groove binding modes, and to obtain by curve fitting association constants K. These show acceptable agreement if derived from several independent NMR signals. Less reliable information is obtained from selected UV titrations, from DNA melting differences, from calorimetric measurements and from affinity comparison to polyamines, the latter being based on a fluorescence assay with ethidium bromide. In contrast to literature expectations the naphthalene-shaped ligands show similar affinities irrespective of the presence of nitrogen atoms, or even of charges within the aromatic system. Quinolinium and naphthalene derivatives only intercalate if they bear a positively charged side chain, and then with similar binding constants. Comparison of all systems as well as salt effects demonstrate that the binding can be quantified with additive contributions from salt bridges of the ammonium centres in the side chains, and from the stacking effects of the aromatic parts. There is no evidence for non-classical intercalation by partial insertion between the nucleobases, nor for any intercalation of phenyl units.