Betaine is an osmolyte present in high concentrations in renal medullary cells. Betaine and other organic osmolytes, such as glycerophosphorylcholine, myo-inositol, and sorbitol, have been shown to increase in concentration during antidiuresis when the inner medullary extracellular osmolality rises. Its concentration may increase in renal cells either by betaine uptake or by choline metabolism to betaine. These studies measured the uptake of (14C)betaine into cortical, outer medullary and inner medullary slices from rat kidney. The tissue-to-medium ratio of (14C) betaine increased with increasing osmolality up to 450 mosmol/kg in outer medullary and inner medullary slices, but not in cortical slices. Betaine uptake increased when the osmolality was raised with NaCl or mannitol, but not with urea. When LiCl was substituted for NaCl in a medium of 300 mosmol/kg, there was significant inhibition of betaine uptake, although the tissue-to-medium ratios remained greater then unity. Thus, increases in osmolality stimulate betaine uptake in rat renal medullary slices and this uptake occurs by both sodium-dependent and sodium-independent betaine transport.