We have examined the effects of l -thyroxine (T 4 ) on the activation of signal transducer and activator of transcription 3 (STAT3) and on the STAT3-dependent induction of c-Fos expression by epidermal growth factor (EGF). T 4 , at a physiological concentration of 100 nM, caused tyrosine phosphorylation and nuclear translocation (i.e. activation) of STAT3 in HeLa cells in as little as 10–20 min. Activation by T 4 of STAT3 was maximal at 30 min (15±4-fold enhancement; mean±S.E.M.) in 18 experiments. This effect was reproduced by T 4 –agarose (100 nM) and blocked by CGP 41251, genistein, PD 98059 and geldanamycin, inhibitors of protein kinase C (PKC), protein tyrosine kinase (PTK), mitogen-activated protein kinase (MAPK) kinase and Raf-1 respectively. Tyrosine-phosphorylated MAPK also appeared in nuclear fractions within 10 min of treatment with T 4 . In the nuclear fraction of T 4 -treated cells, MAPK immunoprecipitate also contained STAT3. The actions of T 4 were similar in HeLa and CV-1 cells, which lack thyroid hormone receptor (TR), and in TR-replete skin fibroblasts (BG-9). T 4 also potentiated the EGF-induced nuclear translocation of activated STAT1α and STAT3 and enhanced the EGF-stimulated expression of c-Fos. Hormone potentiation of EGF-induced signal transduction and c-Fos expression was inhibited by CGP 41251, geldanamycin and PD 98059. Therefore the non-genomically induced activation by T 4 of STAT3, and the potentiation of EGF by T 4 , require activities of PKC, PTK and an intact MAPK pathway.