Renal osmolyte concentrations are reduced during reflow following ischemia. Osmolyte decreases may follow oxygen depletion or loss of extracellular osmolality in the medulla. Image-guided volume-localized magnetic resonance (MR) microspectroscopy was used to monitor regional osmolytes during hyposmotic shock and hypoxia in the intact rat kidney. Alternate spectra were acquired from 24-µl voxels in cortex and medulla of the isolated perfused kidney. There was a progressive decrease in the combined betaine-glycerophosphorylcholine (GPC) peak intensity of 21% in cortex and 35% in medulla of normoxic kidneys between 60 and 160 min after commencing perfusion. Hypoxia had no significant effect on the betaine-GPC peak intensity in cortex or medulla, despite a dramatic reduction in tubular sodium, potassium, and water reabsorption. The results suggest that cortical and medullary intracellular osmolyte concentrations depend on osmotically regulated channels that are insensitive to oxygen and dissociated from the oxygen-dependent parameters of renal function, the fractional excretion of sodium, the fractional excretion of potassium, and urine-to-plasma inulin concentration ratio.