The synthesis of a novel class of exocyclic bis- and tris-3,2-hydroxypyridinone (HOPO) chelators built on N-2 and N-3 aza-macrocyclic scaffolds and the thermodynamic solution characterization of their complexes with Fe(III) are described. The chelators for this study were prepared by reaction of either piperazine or N,N',N''-1,4,7-triazacyclononane with a novel electrophilic HOPO iminium salt in good yields. Subsequent removal of the benzyl protecting groups using HBr/acetic acid gave bis-HOPO chelators N-2(etLH)(2) and N-2(prLH)(2), and tris-HOPO chelator N-3(etLH)(3) in excellent yields. Solution thermodynamic characterization of their complexes with Fe(III) was accomplished using spectrophotometric, potentiometric, and electrospray ionization-mass spectrometry (ESI-MS) methods. The pKa's of N-2(etLH)(2), N-2(prLH)(2), and N-3(etLH)(3), were determined spectrophotometrically and potentiometrically. The Fe(III) complex stability constants for the tetradentate N-2(etLH)(2) and N-2(prLH)(2), and hexadentate N-3(etLH)(3), were measured by spectrophotometric and potentiometric titration, and by competition with ethylenediaminetetraacetic acid (EDTA). N-3(etLH)(3) forms a 1:1 complex with Fe(III) with log beta(110) = 27.34 +/- 0.04. N-2(prLH)(2) forms a 3:2 L:Fe complex with Fe(III) where log beta(230), = 60.46 +/-0.04 and log /3110 = 20.39 +/- 0.02. While N2(etLH)2 also forms a 3:2 L:Fe complex with Fe(III), solubility problems precluded determining log beta(230); log film was found to be 20.45 +/- 0.04. The pFe values of 26.5 for N-3(etLH)(3) and 24.78 for N-2(prLH)(2) are comparable to other siderophore molecules used in the treatment of iron overload, suggesting that these hydroxypyridinone ligands may be useful in the development of new chelation therapy agents.