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Glycerophosphorylcholine, inositol, and sorbitol were measured in rat kidney homogenates and tubules from inner medulla and papilla by enzymatic spectrophotometric techniques. Organic osmolytes exhibited their highest concentrations in the papillary tip. In contrast to glycerophosphorylcholine and sorbitol, inositol was of similar high concentrations in inner and outer medulla. Freshly prepared inner medullary tubules maintained tissue osmolyte concentrations under control, antidiuretic, and furosemide diuretic conditions. When tubules were incubated in vitro over 90 min, tubular organic osmolyte concentrations decreased as a function of extracellular NaCl, but not urea concentrations. Organic osmolyte disappearance from cells was quantitatively recovered from the medium. In contrast, medium lactate dehydrogenase activity did not rise in parallel and tubular ATP remained constant. Glucose up to a concentration of 200 mM increased tubule and medium sorbitol. The results obtained indicate that glycerophosphorylcholine, sorbitol, and inositol rapidly adapt their intracellular concentrations to extracellular NaCl osmolality by a change in tubular plasma membrane permeability. In addition sorbitol levels are regulated by the extracellular glucose concentration.