The effect of thyroid hormones on nuclear RNA synthesis was studied in vitro. Results indicated that thyroid hormones added to cell-free rat liver homogenates, in the presence of IMP as a precursor of purine nucleotides and necessary substrates, stimulated orotic acid-6-¹⁴C incorporation into nuclear RNA. The stimulatory effect was observed with 10⁻⁹ M triiodo-L-thyronine (L-T₃ )and with 10⁻⁵ M thyroxine. Thyroid hormones had no effect on the incorporation of UTP-³H into nuclear RNA which is evidence that the primary site of action of the hormones is in the cytoplasm, i.e., the conversion of IMP to AMP and to GMP. The net effect is to maintain a high adenine nucleotide/guanine nucleotide ratio. The incorporation of UTP-³H into nuclear RNA was enhanced when the ATP/GTP ratio was greater than one as compared to that when the ratio was one, and it was inhibited when the ratio was less than one. The relative concentrations of the purine nucleotides had a profound effect on nuclear RNA synthesis. DNase and actinomycin D inhibited nuclear RNA synthesis induced by a high ATP/GTP ratio. Based on these results, it was proposed that thyroid hormones regulate the synthesis of AMP and GMP from IMP and maintain a high adenine nucleotide/ guanine nucleotide ratio which enhances the synthesis of a specific RNA. The significance of the maintenance of a high adenine nucleotide/guanine nucleotide ratio and the consequences of the synthesis of a specific RNA were discussed. / Master of Science