Thyrotropin (TSH) injected intraperitoneally caused a significant rise in the activity of L-ornithine decarboxylase of rat thyroid within 4 hr. TSH-releasing hormone (TRH) also increased the activity of this enzyme in a dose-related manner and of putrescine-activated S-adenosyl-L-methionine decarboxylase, probably through the increased secretion of TSH from the pituitary. Administration of methylthiouracil (MTU) in the drinking water to rats resulted in an increase of these two enzymes in the thyroid. Maximal activation was observed 4 days after the initiation of MTU treatment for both enzymes. The intracellular level of putrescine and spermidine increased during the rapid phase of thyroid enlargement caused by MTU treatment. The spermine concentration, however, was relatively constant during the treatment. The RNA/DNA ratio followed a pattern very similar to that exhibited by the spermidine/spermine ratio. These results indicate that TSH stimulates the biosynthesis of polyamines by increasing the activity of the decarboxylases in the thyroid. The findings further suggest that polyamines play a role in the regulation of thyroid growth and nucleic acid metabolism.