The purpose of this study was to elucidate the mechanism by which thyroid hormone alters urea synthesis. A set of three experiments was investigated in three groups of rats given 6-propyl-2-thiouracil (a thyroid inhibitor) without triiodothyronine treatment, treated with 6-propyl-2-thiouracil plus triiodothyronine or neither 6-propyl-2-thiouracil nor triiodothyronine (control). We attempted to determine whether the concentration of ornithine and N-acetylglutamate regulated urea synthesis and whether activities of two ornithine-catabolizing enzymes accounted for changes in ornithine concentration. Urinary excretion of urea and the liver concentration of N-acetylglutamate and ornithine in rats given 6-propyl-2-thiouracil plus triiodothyronine were significantly lower than in rats given 6-propyl-2-thiouracil alone. The liver concentration of N-acetylglutamate was correlated to urea excretion (r = 0.911, P less than 0.001). The activities of carbamylphosphate, synthetase, ornithine aminotransferase and ornithine decarboxylase in liver of the group treated with 6-propyl-2-thiouracil alone were significantly lower than those of the 6-propyl-2-thiouracil plus triiodothyronine-treated group. The results suggest that a higher liver concentration of N-acetylglutamate and ornithine in the hypothyroid (6-propyl-2-thiouracil only) rats is likely to stimulate urea synthesis. The thyroid hormone-induced increase in activities of ornithine catabolizing enzymes may be primarily responsible for changes in ornithine concentration.