The binding of the dye cations acriflavine AF, tetramethylacriflavine TMAF and acridine orange AO (scheme of structures) to calf thymus DNA has been investigated by means of absorption spectroscopy, Table I. In order to avoid dye association we used very low dye concentrations and sufficiently high DNA concentrations. In this case we got linear Scatchard isotherms. The formal Scatchard binding constant K strongly depends on the salt concentration Cs (S = NaCl) of the solution and the temperature T (278 - 303 K), K (C S , T). The average value of binding sites per mononucleotide is n = 0.17. It is independent of the dye species and of C S and T. The value of r (bound dye cations per mononucleotide) diminishes with growing salt concentration C S (C S ≲ 1 ᴍ). At sufficiently high salt concentrations r is approximately constant (C S ≳ 1 ᴍ). Obviously there are two types of binding of the dye cations to DNA even in the domain of linear Scatchard isotherms. They can be distinguished experimentally with the competitive salt effect. To describe r(C S ,T ) or K (C S , T) we used a simple model with three equilibria: 1. Noncompetitive binding 1 (intercalation) of dye cations to n 1 C N binding sites (C N = concentration of mononucleotides), equilibrium constant K 1 . 2. Competitive binding 2 (external binding) of dye cations to n 2 C N binding sites, equilibrium constant K 2 . In contrast to type 1 binding, the dye cations in type 2 binding can be replaced by metal cations M of S (M = Na ⊕ ) at sufficiently high salt concentrations C S . 3. Competitive binding 3 of M to the same sites of 2 and the dye cations as competitor, equilibrium constant K 3 . The model agrees very well with the experiments on the condition n 1 = n 2 = n. Therefore the dye can be bound to one of the n C N binding sites either non-competitively or competitively. Type 1 and type 2 binding exclude one another at the same binding site in the domain of linear Scatchard plots. The binding constants K i (i = 1, 2, 3) have been determined by means of the competitive salt effect, Table II. They only are T dependent. From K(T) i we got the binding enthalpies ΔH i 0 and binding entropies ΔS i 0 , Table III. AF and AO cations are bound non-competitively and competitively, TMAF only competitively. In comparison with AF or AO the competitive binding of TMAF is much weaker. In the case of AF and AO K 1 is approximately one power of ten smaller than K 2 , K 1 ⪡ K 2 ! The binding enthalpies of the non-competitive and the competitive binding are nearly equal, ΔH 1 0 ≅ ΔH 2 0 . Therefore the difference in the binding constants K 1 , K 2 can be attributed to the difference in the binding entropies, ΔS 1 0 ⪡ ΔS 2 0 . Thermodynamically type 2 binding (external binding) is preferred to type 1 binding (intercalation). The binding enthalpy of Na ⊕ to DNA is in all cases nearly zero, ΔH 3 0 ≅ 0. Only the increase of entropy S 3 0 > 0 enables binding 3. From the thermodynamic data follows that type 1 and type 2 binding of AF and AO are produced by electrostatic and hydrophobic interaction which are intensified by hydrogen bonding. In contrast to this the weaker competitive binding of TMAF is caused by electrostatic and hydrophobic interaction only. Our investigations agree with former works on ethidium bromide E and tetramethylethidium bromide TME (scheme of structures, Tables II and III). They are consistent with the assignment non-competitive binding = intercalation, competitive binding = external binding.