The purpose of this study was to find whether or not the ornithine transport into mitochondria regulated urea synthesis when the thyroid status is 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 receiving neither PTU nor T3 (control). The urinary excretion of urea, liver concentration of ornithine and ornithine transport into isolated hepatic mitochondria in rats given PTU+T3 were significantly lower than in rats given PTU alone. Ornithine transport was significantly inhibited by the addition of lysine specifically. This response was achieved well within the physiological concentration of lysine. Compared with rats given PTU without T3 treatment, the liver concentration of lysine was significantly higher in rats treated with PTU+T3 and control rats. Ornithine transport into hepatic mitochondria was closely correlated with the excretion of urea. The results suggest that the greater ornithine transport in the hypothyroid (PTU alone) rats is likely to stimulate urea synthesis. A thyroid hormone-induced increase in lysine concentration may be at least partly responsible for the changes in ornithine transport into mitochondria.