In vivo sodium-23 and hydrogen-1 magnetic resonance (MR) imaging and spectroscopy of the rat brain during infusion of the shift reagent thulium DOTP5- (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra[methylene phosphonate] was performed to assign the various peaks observed during infusion and to evaluate the shift reagent in discriminating tissue compartments. Na-23 spectra collected during the infusion showed two shifted peaks that were assigned to intravascular Na+ and extracellular muscle Na+, respectively, and one unshifted peak assigned to intra- and extracellular brain Na+ and cerebrospinal fluid Na+. These assignments were validated with H-1 and Na-23 MR imaging and Na-23 chemical shift imaging (CSI). The H-1 and Na-23 images showed that a surface coil placed on a rat head can detect a substantial amount of signal from muscle surrounding the skull. Na-23 CSI spectra from successive 1-mm-thick coronal sections indicated that the shift reagent did not cross the blood-brain barrier. The study also showed that bulk susceptibility shifts are quite small with Tm-DOTP5-. This reagent may be useful in determining compartmental Na+ concentrations and blood flow kinetics in brain and in examining the integrity of the blood-brain barrier.