We report the synthesis of two pyclen-based regioisomer ligands (pyclen = 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene) functionalized with picolinic acid pendant arms either at positions 3,9-pc2pa (L5) or 3,6-pc2pa (L6) of the macrocyclic fragment. The ligands were prepared by the regiospecific protection of one of the amine nitrogen atoms of the macrocycle using Boc and Alloc protecting groups, respectively. The X-ray structure of the Gd(III) complex of L5 contains trinuclear [(GdL5)(3) (H2O)(3)](3+) entities in which the monomeric units are joined by mu(2)-eta(1):eta(1) -carboxylate groups. However, the H-1 and Y-89 NMR spectra of its Y(III) analogue support the formation of monomeric complexes in solution. The Tb(III) complexes are highly luminescent, with emission quantum yields of up to 28% for [TbL5](+). The luminescence lifetimes recorded in H2O and D2O solutions indicate the presence of a water molecule coordinated to the metal ion, as also evidenced by the H-1 relaxivities measured for the Gd(III) analogues. The Gd(III) complexes present very different exchange rates of the coordinated water molecule (k(ex)(298)= 87.1 X 10(6) and 1.06 X 10(6) s(-1) for [GdL5](+) and [GdL6](+) , respectively). The very high water exchange rate of [GdL5](+) is associated with the steric hindrance originating from the coordination of the ligand around the water binding site, which favors a dissociatively activated water exchange process. The Gd(III) complexes present rather high thermodynamic stabilities (log K-GdL = 20.47 and 19.77 for [GdL5](+) and [GdL6](+), respectively). Furthermore, these complexes are remarkably inert with respect to their acid-assisted dissociation, in particular the complex of L5.