A simple high performance liquid chromatography (HPLC) method of separating and quantitating the predominant organic solutes of the renal medulla is described. These organic solutes include myo-inositol, glycerophosphorylcholine, sorbitol, betaine, and urea. Other physiologically significant solutes, including glucose and mannitol, can be separated and quantitated concurrently with this method. With the use of this technique, the organic solutes of the rabbit kidney were determined. No new organic compounds were detected by HPLC that could significantly contribute to intracellular osmolality of the medulla. The values for the organic solutes already described were similar to those obtained by more complicated and limited approaches such as classical enzymatic techniques, ion electrodes, nuclear magnetic resonance spectroscopy, and gas chromatography-mass spectroscopy.