New N4O2-donor acyclic chelators 2-[[2-[2-(3,5-dimethylpyrazol-1-yl)ethyl-[2-[(2-hydroxyphenyl)methylamino]ethyl]amino]ethylamino]methyl]phenol (H2Lpz*,NH) and 2-[[2-[2-[(2-hydroxyphenyl)methylamino]ethyl-(2-pyridylmethyl)amino]ethylamino]methyl]phenol (H2Lpy,NH) were obtained upon introduction of pyridyl or pyrazolyl coordinating units at the central nitrogen atom of diethylenetriamine (dien) and by functionalization of its terminal amines with phenol groups. The coordination behavior of H2Lpz*,NH and H2Lpy,NH was evaluated towards natGa/67Ga, aiming to assess their suitability to obtain Ga(III) chelates relevant for biomedical applications. Single crystal X-ray diffraction analysis of the complexes [GaLpz*,NH](ClO4) and [GaLpy,NH](ClO4), confirmed the presence of N4O2-hexadentate chelators with the phenoxide groups coordinated cis relatively to the pyridyl/pyrazolyl arms. Unlike [GaLpz*,NH](ClO4), [GaLpy,NH](ClO4) exists in solution as a mixture of isomers, as confirmed by several NMR techniques. The corresponding radiocomplex [67GaLpy,NH]+ was obtained smoothly in almost quantitative radiochemical yield, contrarily to [67GaLpz*,NH]+ that seems to be (hemi)labile under the same conditions. [67GaLpy,NH]+ presents a high in vivo stability and a favourable biodistribution profile in mice. The imine congeners 2-[(E)-2-[2-(3,5-dimethylpyrazol-1-yl)ethyl-[2-[(E)-(2-hydroxyphenyl)methyleneamino]ethyl]amino]ethyliminomethyl]phenol (H2Lpz*,C=N) and 2-[(E)-2-[2-[(E)-(2-hydroxyphenyl)methyleneamino]ethyl-(2-pyridylmethyl)amino]ethyliminomethyl]phenol (H2Lpy,C=N) were also evaluated but they did not form complexes compatible with biomedical applications owing to their high reactivity.