Recent reports have claimed a superior performance of HEPES buffer in comparison to alternative buffer systems for 67/68 Ga labeling in aqueous media. In this paper we report spectroscopic (1H and 71 Ga NMR), radiochemical, mass spectrometry and theoretical modeling studies on the Ga3+/HEPES system (HEPES = N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) performed with the aim of elucidating a potential contribution of HEPES in the 68/67 Ga radiolabeling process. Our results demonstrate that HEPES acts as a weakly but competitive chelator of Ga3+ and that this interaction depends on the relative Ga3+: HEPES concentration. A by-product formed in the labeling mixture has been identified as a [68 Ga]Ga(HEPES) complex via chromatographic comparison with the nonradioactive analog. The formation of this complex was verified to compete with [68 Ga]Ga(NOTA) complexation at low NOTA concentration. Putative chelation of Ga3+ by the hydroxyl and adjacent ring nitrogen of HEPES is proposed on the basis of 1H NMR shifts induced by Ga3+ and theoretical modeling studies. Copyright © 2013 John Wiley & Sons, Ltd.