Gabaculine, a potent suicide inhibitor of ornithine aminotransferase (OAT), at a dose of 50 mg/kg inhibited this enzyme in mouse tissues and dramatically increased tissue ornithine concentrations, whether or not arginine was present in the diet. Thus even under arginine deprivation there is catabolism of ornithine which involves OAT. This was confirmed by administration of [14C]ornithine to arginine-deprived mice. Gabaculine (3-amino-2,3-dihydrobenzoic acid) drastically decreased the release of 14CO2 and increased the radioactivity in the basic amino acids in the tissues. When [1-14C]glutamate was injected into mice deprived of arginine, a significant amount of radioactivity was recovered in tissue ornithine and arginine, and gabaculine decreased this labelling by about two-thirds, indicating that ornithine was synthesized in vivo from glutamate via OAT. In addition, we failed to detect in liver and small intestine alpha-N-acetylornithine, N-acetylglutamate kinase or N-acetylornithine aminotransferase, which are obligatory components of a potential route of ornithine synthesis from N-acetylglutamate. Our results indicate that at least 45 mumol of ornithine was synthesized and catabolized daily via OAT in the mouse deprived of arginine.