The purpose of this study was to find whether the regulation of urea synthesis was mediated through the activation of N-acetyglutamate synthesis by ornithine when the thyroid status was manipulated. Experiments were done on three groups of rats, given 6-propyl-2-thiouracil (PTU, a thyroid inhibitor) without triiodothyronine (T3) treatment, treated with PTU + T3, or neither PTU nor T3 (control). Urinary excretion of urea and the liver concentrations of ornithine and N-acetylglutamate in rats given PTU + T3 were significantly lower than in rats given PTU alone. The liver concentration of N-acetylglutamate was correlated with the liver concentration of ornithine (r = 0.920, p < 0.001). Ornithine administration (0.5 mmol/100 g body wt) elevated the liver concentration of N-acetylglutamate in all three groups. The results suggest that the greater concentration of ornithine in the hypothyroid (PTU alone) rats is likely to increase the N-acetylglutamate concentration in liver and stimulate urea synthesis.