Cells generally adapt to long-term hyperosmolality by accumulating compatible organic osmolytes, thereby helping to normalize both volume and intracellular inorganic ion concentration. When organic osmolytes are accumulated, as in renal inner medullary cells, it is the sum of their concentrations that is theoretically important. In effect, when one organic osmolyte rises, the others generally fall to maintain their sum approximately constant. The present study addresses the mechanism controlling betaine accumulation. Hypertonicity induces accumulation of betaine, sorbitol, inositol, and other organic osmolytes in PAP-HT25 cells, a line derived from rabbit renal papilla. Hypertonicity increases the betaine transporter expression in these cells. To obtain a specific probe for betaine transporter mRNA, we cloned from PAP-HT25 cells a cDNA that encodes the full protein. We then examined the effect of betaine, sorbitol, and inositol on betaine transporter mRNA abundance. Increased accumulation of any of these three organic osmolytes reduces betaine transporter mRNA. We previously observed similar results for aldose reductase, the enzyme responsible for osmotically regulated sorbitol accumulation. We conclude that the accumulation of organic osmolytes regulates betaine transporter gene expression. Because the aldose reductase gene is controlled in a similar fashion, we surmise that the two genes share a common signal for induction.