Effect of chronic methylthiouracil (MTU) treatment on the thyroid arginase activity and thyroidal concentration of arginine, ornithine and other amino acids was studied in the rat. The activity of thyroid arginase increased significantly at 15 days of MTU treatment and the elevated enzyme activity was reduced to normal by l-thyroxine (T4) injection. The thyroidal concentration of polyamines was increased by MTU and decreased by T4 with the exception of spermine. The thyroidal concentration of arginine and lysine, a substrate and an inhibitor for arginase respectively decreased significantly, while that of ornithine remained unchanged after MTU treatment. T4 injected to MTU-pretreated rats restored the decreased arginine concentration to normal. These results suggest that ornithine supply for polyamine biosynthesis is regulated by the level of both arginase and lysine in the thyroid.