Three new macrocycles with a 26-membered interior ring size and four NH-based binding moieties have been prepared. The three macrocycles were shown, both by computational modelling as well as 1D-ROESY experiments, to exhibit significantly different degrees of preorganization, originating from different degrees of rigidity within their macrocyclic structures. Binding studies with anions of varying size (Cl -, H2PO4 -, and H2P2O7 2-) revealed very significant differences between the macrocycles. The highly flexible macrocycle 3 favors intramolecular hydrogen bonding over interactions with anions. The rigid macrocycle 1 exhibits, not unexpectedly, poor interaction with Cl -since the cavity was too large, but showed good interactions with H2PO4 -and H2P2O7 2-. Macrocycle 2 exhibits the best anion affinity for all three anions, most likely due to the combination of its preorganization and its ability to adapt its conformation more easily than macrocyclic receptor 1. Furthermore, in addition to four acylhydrazone NH H-bond donors, it has six aromatic protons pointing into the binding cavity. X-ray crystallography of complexes of macrocycle 2 with the three anions confirmed its ability to bind these different anions, of which H2PO4 -was bound with a 1:2 host to guest stoichiometry.