Epidermal growth factor (EGF) and TGFalpha share the same plasma membrane receptor. In the present studies in HeLa cells, both EGF and TGFalpha caused MAPK (ERK1/2) activation and expression of the immediate-early gene c-fos. Thyroid hormone (T(4)) nongenomically enhanced EGF- and TGFalpha-induced MAPK activation. This T(4) action was duplicated by T(4)-agarose and blocked by tetraiodothyroacetic acid, which inhibits binding of T(4) to plasma membranes. TGFalpha-induced MAPK activation was potentiated by 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP) but not 8-chloro-cyclic adenosine monophosphate. TGFalpha, T(4), and 8-Br-cAMP each caused protein kinase A (PKA) II serine phosphorylation, whereas phosphorylation of PKA-II was not seen in cells treated with EGF or 8-chloro-cyclic adenosine monophosphate. In a PKA activity assay, the enzyme was stimulated by T(4), EGF, and TGFalpha; T(4) enhanced the effect of TGFalpha but not that of EGF. T(4), although it potentiated c-fos gene expression in EGF-treated cells, suppressed this effect in cells treated with TGFalpha. Cells exposed to 8-Br-cAMP also inhibited TGFalpha-stimulated c-fos expression. Studies of cell proliferation indicated that T(4) potentiated EGF action but inhibited that effect in TGFalpha-treated cells. The disparate effects of T(4) on actions of EGF and TGFalpha, which share the same cell surface receptor, are mediated by hormone phosphorylation and activation of PKA-II.