The regulation of a gene, designated spot 14, which is rapidly induced in rat liver in response to 3, 5, 3′-triiodo-L-thyronine (T 3) was studied as a model for exploring the molecular basis of thyroid hormone action. The time course of induction of the nuclear precursor to spot 14 mRNA after intramuscular injection of T 3 displayed a very short lag period of between 10 and 20 min. The rapidity of this effect suggests that the induction in gene expression occurs as a primary response to the hormone-receptor interaction. The protein synthesis inhibitor cycloheximide injected 15 min before T 3 completely blocked the accumulation of nuclear precursor RNA 30 min after T 3 treatment. Emetine, an inhibitor of protein synthesis which acts by a different mechanism than cycloheximide, also blocked the induction of the spot 14 nuclear precursor RNA. The increased rate of spot 14 gene transcription observed after T 3 treatment, as measured by nuclear run-on assay, was similarly completely abolished in the presence of cycloheximide. In addition, ongoing protein synthesis was required for maintaining spot 14 nuclear precursor RNA at induced levels in animals previously treated with T 3. On the other hand, cycloheximide had no effect on T 3 uptake or binding to the nuclear receptor during the 45-min time frame studied. The paradox of the rapid kinetics of induction and the requirement of ongoing protein synthesis may be explained by a protein with an extremely short half-life which is necessary for T 3 induction of the spot 14 gene.