Multiple data suggest a relationship between thyroid hormone (triiodothyronine (T3)) and carcinogenesis. Studies on breast cancer have been inconclusive, suggesting contradictory effects of thyroid status and diseases. Recently, we reported that expression of the extracellular matrix glycoprotein tenascin-C is modulated by T3 during rat brain development. Because tenascin-C has been reported to have growth-, motility-, and angiogenic-promoting activities and to become upregulated during tumorigenesis in breast carcinoma and stromal cells, we analyzed the effects of T3 on tenascin-C expression in mammary epithelial cells. In this study, we showed that tenascin-C RNA expression was inhibited by T3 in normal un-transformed EpH4 mouse mammary epithelial cells expressing appropriate receptors. T3's action appeared to be due to a decreased half-life of the tenascin-C mRNA, with a maximum effect (85% at 100 nM) 48 h after addition. T3 also downregulated tenascin-C in the human mammary tumor cell line SKBR-3, which expresses endogenous thyroid receptors. Immunoprecipitation experiments confirmed that tenascin-C protein content was also decreased by T3 in EpH4 cells (70% reduction at 100 nM). Dexamethasone had a similar inhibitory effect (70% at 100 nM), whereas estradiol, the antiestrogen ICI 164,384, progesterone, and all-trans retinoic acid did not alter tenascin-C expression. Our data demonstrate an inhibitory action of T3 on tenascin-C expression in mammary epithelial cells that may play a role in the physiological regulation of this gene and in neoplastic processes.