Guanidinoacetic acid (GAA), a precursor of creatine, is an essential substrate for muscle energy metabolism. GAA is formed from arginine and glycine by glycine-amidinotransferase (GAT) mainly in the kidney. But mechanism of GAA synthesis in the kidney has not been defined. The author isolated renal tubules from rats and incubated them with various substrates. GAA was separated by HPLC and measured fluorometrically after reacting with 9, 10-phenanthrenequinone. The following results were obtained: 1. GAA synthesis in the isolated renal tubules was recognized without addition of any substrate. 2. A remarkable increase of the amount of GAA was observed in the presence of glycine and several amidine donors, such as arginine and canavanine. 3. The amount of GAA tended to increase until 3 hours. 4. D.L-norvaline and ornithine suppressed GAA synthesis in renal tubules. 5. Methionine suppressed GAA synthesis. 6. Creatine suppressed GAA synthesis. Negative feedback control of GAA synthesis by creatine was recognized in vitro. 7. GAA synthetic activity was not recognized after the addition of hydroxyurea, citrulline and argininosuccinic acid in the medium. 8. Low concentrations of methylguanidine and guanidinosuccinic acid decreased the amount of GAA. Concentrations used in the experiment were similar to those in uremic serum.