Carnitine biosynthesis was studied in man and rat. Three healthy adult men were given intravenous injections of 1 mCi of [methyl-3H]epsilon-N-trimethyl-L-lysine, a precursor of carnitine. Labeled metabolites of this compound were monitored in serum and urine at 2, 6, 12, 24, and 48 h. At least nine radioactive metabolites were detected. For each collecton period, the specific activity of urinary carnitine exceeded the average serum specific activity. In man, the amount of labeled carnitine in urine was 2 to 8 times greater than labeled gamma-butyrobetaine (the immediate precursor of carnitine). In similar experiments in rats (intravenous injection of 0.1 mCi of [methyl-3H]epsilon-N-trimethyl-L-lysine), the specific activity of carnitine in urine was always lower than the corresponding average specific activity in serum. Between 0 and 2 h after administration of labeled precursor, the animals excreted large amounts of labeled gamma-butyrobetaine but little labeled carnitine. Significant gamma-butyrobetaine, 2-oxoglutarate dioxygenase (EC 1.14.11.1) activity was found in human kidney but this activity was absent in rat kidney. The results indicate that in man and rat the kidney accumulates intravenously administered [methyl-3H]epsilon-N-trimethyl-L-lysine. This compound is metabolized predominantly to gamma-butyrobetaine in rat kidney and to carnitine in human kidney. In both species, the synthesized products are at least partially leaked (either by secretion or by passive diffusion down a concentration gradient) into the renal tubular lumen from which they are either reabsorbed into the circulation for distribution to other tissues or excreted.