AbstractUnder normal conditions, high sodium (Na⁺) in extracellular (Na⁺_e) and blood (Na⁺_b) compartments and low Na⁺ in intracellular milieu (Na⁺_i) produce strong transmembrane (ΔNa⁺_mem) and weak transendothelial (ΔNa⁺_end) gradients respectively, and these manifest the cell membrane potential (V_m) as well as blood–brain barrier (BBB) integrity. We developed a sodium (²³Na) magnetic resonance spectroscopic imaging (MRSI) method using an intravenously-administered paramagnetic polyanionic agent to measure ΔNa⁺_mem and ΔNa⁺_end. In vitro ²³Na-MRSI established that the ²³Na signal is intensely shifted by the agent compared to other biological factors (e.g., pH and temperature). In vivo ²³Na-MRSI showed Na⁺_i remained unshifted and Na⁺_b was more shifted than Na⁺_e, and these together revealed weakened ΔNa⁺_mem and enhanced ΔNa⁺_end in rat gliomas (vs. normal tissue). Compared to normal tissue, RG2 and U87 tumors maintained weakened ΔNa⁺_mem (i.e., depolarized V_m) implying an aggressive state for proliferation, whereas RG2 tumors displayed elevated ∆Na⁺_end suggesting altered BBB integrity. We anticipate that ²³Na-MRSI will allow biomedical explorations of perturbed Na⁺ homeostasis in vivo.