Thyroid hormone receptors (TRs) are members of the ligand-inducible transcription factor superfamily. The two major functional TRs (a1 and b1) have different spatial and temporal expression patterns and specific physiological functions for these isoforms are now starting to emerge. By expressing these TR isoforms individually in Swiss 3T3 fibroblasts, we found that TRb1 expression, in the absence of hormone, provokes a proliferation arrest in G0/G1, lengthening the cycling time. Upon serum stimulation TRb1-expressing cells showed a marked delay in the induction of cyclins D and E, in the phosphorylation of retinoblastoma protein, and in the activation of cyclindependent kinase 2, accompanied by increased levels of cyclin-dependent kinase inhibitor p27 Kip1 . Accordingly, serum-stimulated E2F-1 transcriptional activity was repressed by TRb1 in transient transfection experiments. Analysis of the receptor domains required for this effect confirmed that there is no need for a functional ligandbinding domain while the DNA-binding domain is essential. In this work, we demonstrate for the first time that TRb1 participates in the molecular mechanisms that control cell proliferation. The unliganded TRb1 impairs the normal induction of the G1/S cycle regulators preventing progression into the S phase.