A thorough thermodynamic isothermal titration calorimetric analysis of allosteric and chelate cooperativity effects in divalent crown ether–ammonium complexes is combined with density functional theory calculations including implicit solvent on one hand and large-scale molecular dynamics simulations with explicit solvent molecules on the other. The complexes studied exhibit binding constants up to 2 • 106 M−1 with large multivalent binding enhancements and thus strong chelate cooperativity effects. Slight structural changes in the spacers, i.e. the exchange of two ether oxygen atoms by two isoelectronic methylene groups, cause significantly stronger binding and substantially increased chelate cooperativity. The analysis is rounded up by the examination of solvent effects and allosteric cooperativity. Such a detailed understanding of the binding processes will help efficiently designing and constructing larger supramolecular architecture with multiple, multivalent building blocks.