Renal medullary cells contain high concentrations of "compatible" organic osmolytes, such as myo-inositol, betaine, sorbitol, and glycero-phosphorylcholine. These organic osmolytes accumulate as an osmoregulatory response to the high and variable interstitial NaCl concentration that is part of the urinary concentrating mechanism. Madin-Darby canine kidney (MDCK) cells in culture were previously shown to accumulate myo-inositol and betaine in response to increased NaCl. These organic osmolytes are taken up by sodium-dependent active transport into the cells from the medium. The maximum concentration is not reached until 2-4 days after an increase in medium osmolality. The purpose of this study was to characterize the response to a decrease in medium osmolality of cells that had been grown at a high osmolality. The initial response to decreased osmolality was a rapid, transient efflux of both myo-inositol and betaine from the cells. Efflux was greatest during the first 15 min and resulted in a reduction of cell myo-inositol and betaine by almost 13 and 22%, respectively, after 3 h. Active myo-inositol and betaine influx fell more slowly, reaching a lower limit after approximately 1-2 days. The reduced influx was followed by progressive decrease in cell myo-inositol and betaine to approximately 30% of the initial value after 6 days. Thus, after a decrease in medium osmolality, MDCK cell myo-inositol and betaine fell because of a rapid, transient increase in efflux and a slow, sustained decrease in active influx.