The thyroid hormone receptor (TR) is a member of the nuclear hormone receptor (NHR) superfamily. Members of this superfamily exhibit diverse subcellular distributions and nucleocytoplasmic transport mechanisms. TR exhibits a dual role as a repressor (or activator) in the absence of thyroid hormone (T3), and an activator (or repressor) of transcription in the presence of T3, thus implying constitutive nuclear localisation and persistent contact of TR with DNA. To investigate the complexity of the cellular response to T3, the nucleocytoplasmic distribution of TR, its viral oncogenic variant (v-ErbA), and a DNA-binding mutant (CI22A) were studied, using SDS-PAGE and fluorography to detect radiolabelled proteins extracted from microinjected Xenopus oocytes, and green fluorescent protein (GFP) fusions to study trafficking in transfected NIH/3T3 cells. Surprisingly, after cytoplasmic injection only 39-40% of TRα, TRβ, and C122A was localised to the oocyte nucleus, increasing to 45-51 % in response to T3. After nuclear injection, 62% of TRα was retained in the nucleus in the absence of T3 and 85% in the presence, suggesting that more intranuclear binding sites are available for interaction with T3-bound receptor. Only 10% of v-ErbA was localised to oocyte nuclei after cytoplasmic injection, due to association of v-ErbA with hsp90. A similar cytoplasmic subpopulation of v-ErbA/hsp90 in mammalian cells has been previously reported. The distribution of v-ErbA was unaffected by T3. Nuclear accumulation in oocytes is not due to DNA binding alone, since C122A distribution was similar to TR. Interestingly, import of TR and C122A, but not v-ErbA, in oocytes was energy and temperature independent and not inhibited by wheat germ agglutinin. However, export of TRα, TRβ, and C122A was temperature dependent. In contrast to Xenopus oocytes, EGFP-tagged TRα localised entirely to the nuclei in transfected mouse cells, even in energy-depleted and chilled cells, and EGFP-tagged C122A remained extranuclear. Both TRα and EGFP-tagged TRα were shown to be associated with the nuclear matrix in biochemically fractionated NIH 3T3 cells. C122A and EGFP-tagged C122A were not found in the nuclear fractions, confirming the observations in living cells. A universal model for TR localisation is proposed to account for the observations made in the two cell systems. Since nuclear localisation of TR, v-ErbA, and C122A is not constitutive in Xenopus oocytes, this provides a model system for reconstituting regulatory networks affecting nuclear or cytoplasmic retention of TR and its variants in vivo.