Two N-substituted 3-hydroxy-4-pyridinones (1-(3'-aminopropyl)-3-hydroxy-2-methyl-4-pyridinone (L1) and 1-(2'-carboxyethyl)-3-hydroxy-2-methyl-4-pyridinone (L2)) were prepared through one- and three-step reactions, respectively. The pKa values of the ligands and the stability constants of their Ga(III) complexes have been determined. Both the complexes are strongly coordinated to three (O,O) hydroxypyridonate moieties. There is a clear effect of the N-substituents in the lipophilic-hydrophilic balance and in the Ga(III) binding interaction; the acid derivative (L2) has lower lipophilicity but higher chelating strength than the amine derivative (L1). Both chelators are shown to interfere in the typical biological behavior of 67Ga-citrate in mice: L1 enhanced the urinary excretion leading to an increased 67Ga removal from the soft tissue, while L2 induced a lower blood clearance with a pronounced bone uptake mainly at 48 h after injection, thus suggesting that the 67Ga-L2 complex could have potential interest as a bone imaging agent.