Two new tripodal tris(3-hydroxy-4-pyridinone) hexadentate chelators-NTA(BuHP)(3) and NTP(PrHP)(3) (NTA=nitrilotriacetic acid, NTP=nitrilotripropionic acid, HP=hydroxypyridone)-have been developed and studied in solution for their iron and aluminium binding affinity, and also assayed in vivo for their capacity to remove metal from an animal model that is overloaded. These chelators are positional isomers, possessing identical general structures based on aminotricarboxylic acid skeletons attached to three bidentate 3-hydroxy-4-pyridinones (3,4-HPs), but differing in the position of the amide linkage along the chelating "arm". In spite of expected differences in the tripodal ligands, such as acidity and hydrogen-bonding networks, they share important properties, namely, a mild hydrophilic character (log P ca. -1.2 to -1.4) and a strong chelating affinity for Fe and Al (pFe=27.9 and pAl=22.0 for NTA(BuHP)(3); pFe=29.4 and pAl=22.4 for NTP(PrHP)(3)). They also evidenced identical effects on the biodistribution and on the excretion of a radiotracer ((67)Ga) previously administered to mice, as models of iron overload animals. Comparison of the new compounds with reported analogues shows good improvement in terms of solution and in vivo sequestering properties, thus giving support to expectations about their potential clinical application as metal removal agents.