High concentrations of organic solutes are present in the medulla of the antidiuretic kidney. However, their role in and response to acute changes in the diuretic state are unknown. In this study the organic solute content of the renal medulla was determined from extracts with the use of high-performance liquid chromatography following the acute dilution of the medullary interstitium during various forms of diuresis. After acute infusion of saline and furosemide, inner medullary urea, sodium, inositol, sorbitol, and betaine decrease significantly with no change in glycerophosphorylcholine (GPC) content. After diuresis, inner medullary urea and sodium contents eventually returned to control levels, although inositol, sorbitol, and betaine contents still remained low. Addition of antidiuretic hormone to the saline/furosemide infusion gave similar results. In contrast, induction of diuresis from mannitol infusion caused an acute decrease in all 4 organic solutes, whereas glucose infusion caused an acute decrease in all organic solutes except sorbitol. These data demonstrate that a decrease in all four organic solutes can accompany medullary dilution. However, GPC and sorbitol do not decrease when diuresis is induced by furosemide or glucose, respectively. In addition, the recovery of these compounds in a normally functioning kidney after diuresis is much slower than the regeneration of the sodium chloride and urea gradients.