The trimethylamine glycine-betaine is accumulated to high concentrations in medullary cells of mammalian kidneys, whereas betaine synthesis from choline is predominant in the renal cortex. We investigated the contribution of renal betaine synthesis to medullary betaine accumulation. De novo synthesis of betaine in situ was accomplished by injecting [14C]choline into the renal artery of male Sprague-Dawley rats. [14C]betaine was measured in the renal cortex and medulla, as well as in serum and urine samples. Betaine concentration in the cortex decreased from 3.5 +/- 1.3 at 5 min to 0.4 +/- 0.2 nmol/mg protein at 60 min, but it increased from 1.4 +/- 0.1 to 2.5 +/- 0.6 nmol/mg protein in the medulla. Serum and total urine [14C]betaine increased from 2.7 +/- 1.3 and 0.9 +/- 0.1 nmol/ml at 5 min to 5.3 +/- 0.3 and 2.1 +/- 0.4 nmol/ml at 60 min, respectively. Concentrations of newly synthesized betaine were not decreased by the ligation of the hepatic artery and portal vein, suggesting that most [14C]betaine was synthesized in the kidney. Coinjection with 5 mM dimethylamino-ethanol, a choline oxidase inhibitor, and 100 mM cold betaine reduced medullary betaine accumulation by 80 and 76%, respectively. Water deprivation for 60 h increased both cortical and medullary [14C]betaine, whereas furosemide diuresis decreased the medullary [14C]betaine concentration. We concluded that betaine synthesized in the kidney can be accumulated in the medulla and that the medullary concentrations of newly synthesized betaine are closely related to the hydration state of the animal.