Guanidinoacetic acid, a precursor of creatine, is an essential substrate for muscle energy metabolism. Since guanidinoacetic acid has been reported to be synthesized from arginine and glycine by glycine amidinotransferase (transamidinase) in kidney homogenates or slices, the purpose of this study was to provide evidence of guanidinoacetic acid synthesis in isolated tubules from rat kidneys, and to clarify the mechanism regulating it. Isolated rat tubules were incubated with various substrates. Guanidinoacetic acid was separated by high performance liquid chromatography and measured fluorometrically. Results obtained were as follows: (1) Guanidinoacetic acid was synthesized from arginine or canavanine and glycine in isolated rat tubules. (2) D,L-Norvaline, ornithine and methionine suppressed guanidinoacetic acid synthesis. (3) Creatine suppressed guanidinoacetic acid synthesis, i.e. creatine was a negative feedback inhibitor of guanidinoacetic acid synthesis in this in vitro system. (4) Guanidinoacetic acid was not synthesized from hydroxyurea, citrulline, argininosuccinic acid or canaline. These data demonstrate that guanidinoacetic acid is synthesized only from arginine or canavanine and glycine, and that the guanidine cycle may not function fully in the rat renal tubule.