Escherichia coli and Staphylococcus typhimurium are known to accumulate betaine by increased transport when extracellular osmolality rises. In the present studies a similar process is demonstrated in mammalian cells. Renal medullary cells contain high concentrations of "compatible" organic osmolytes such as betaine, myo-inositol, sorbitol, and glycerophosphorylcholine. The organic osmolytes occur as an osmoregulatory response to the high and variable interstitial NaCl concentration that is part of the urinary concentrating mechanism. Dog kidney cells in culture (MDCK) were previously shown to accumulate betaine in response to increased extracellular osmolality. We demonstrate here that this accumulation requires the presence of betaine in the medium, and this apparently is a result of uptake of extracellular betaine, rather than synthesis by the cells. MDCK cells have low- and high-affinity sodium-dependent betaine transporters with Km for betaine of approximately 6 and approximately 0.1 mM, respectively. Relative to isotonic controls, sodium-dependent betaine uptake is approximately sevenfold greater in cells chronically exposed (greater than 1 yr) to hypertonic medium (615 mosmol/kg). This is due to an increase in the maximal velocity of sodium-dependent betaine uptake with no apparent change in Km. Cells acutely exposed (1-7 days) to hypertonic medium show increased sodium-dependent betaine uptake, which is maximal after 1 day, then decreases as betaine and other osmolytes accumulate in the cells. Thus the response by which renal cells accumulate betaine following hypertonicity apparently includes an increase in the number (or, less likely, the transport turnover rate) of functioning sodium-dependent betaine transporters.