In order to understand the metabolic abnormalities occurring with renal failure, the biosynthetic pathway of guanidinosuccinic acid (GSA) was investigated in normal rat liver, because GSA is known as a uremic toxin and its synthetic pathway is unclear.After fresh liver was homogenized and centrifuged for 60 min at 100,000×g, the supernatant was fractionated by precipitation with ammonium sulfate. It was found that the activity of GSA biosynthetic enzymes was highest in the fraction between 30% and 40% saturation of ammonium sulfate. The reaction mixture contained potassium phosphate buffer (pH7.4), aspartate, urea, ATP and an ATP-generating system, MnCl(2), and enzyme solution. The optimum pH was 7.4. The activity was strongly accelerated by Mn(++) and Cu(++), while it was completely inhibited by Fe(++). Hydroxyurea, L-canaline, L-canavanine, and L-arginine could not substitute for urea.These results suggest that GSA is not formed by the transamidination of arginine to aspartate as proposed by Cohen, or by way of the guanidine cycles proposed by Natelson. It is possible that GSA is mainly synthesized from urea and aspartate directly in rat liver.