Localization of substrates in membrane proteins is an important but challenging task. In this paper, we show that deuterium electron spin echo envelope modulation spectroscopy ( 2 H ESEEM) combined with site-directed spin labeling is a powerful tool to localize the substrate, histidine-d5, in the ABC transporter HisQMP 2 . Based on a homology model and spin label rotamer analyses, we calculated 2 H ESEEM spectra for eight possible labeling positions close to the putative substrate-binding site. Experimental 2 H ESEEM spectra were determined with spin labels bound either at position 169 of HisM, for which a detectable 2 H ESEEM signal was calculated, or with a spin label bound at position 54 of HisQ as a negative control. The agreement between the calculated and experimental ESEEM spectra provides strong evidence for the histidine located in a binding site primarily liganded by residues of HisM as proposed by the homology model.