p38 mitogen-activated protein kinases (p38-MAPKs) are activated by cytokines, cellular stresses, growth factors, and hormones. We show here that p38-MAPKs are activated upon stimulation by thyroid-stimulating hormone (TSH) or cAMP. TSH caused the phosphorylation of p38-MAPK in Chinese hamster ovary cells stably transfected with the human TSH receptor but not in wild-type Chinese hamster ovary cells. The effect of TSH was fully mimicked by the adenylyl cyclase activator, forskolin, and by a permeant analog of cAMP. The effect of forskolin was reproduced in FRTL5 rat thyroid cells. TSH also stimulated the phosphorylation of MAPK kinase 3 or 6, over the same time scale as that of p38-MAPKs. TSH and forskolin stimulated the activity of the ␣-isoform of p38-MAPK assayed by phosphorylation of the transcription factor ATF2. The activity of MAPKactivated protein kinase-2 was stimulated by TSH and forskolin. This stimulation was abolished by SB203580, a specific inhibitor of p38-MAPKs. The protein kinase A inhibitor H89 inhibited the stimulation of phosphorylation of p38-MAPKs by forskolin, whereas inhibitors of protein kinase C, p70S6k , and phosphatidylinositol 3-kinase were ineffective. Expression of the dominant negative form of Rac1, but not that of Ras, blocked forskolin-induced p38-MAPK activation. Diphenylene iodonium, a potent inhibitor of NADPH oxidase(s), and ascorbic acid, an effective free radical scavenger, suppressed TSH-or forskolin-stimulated p38-MAPK phosphorylation, indicating that the generation of reactive oxygen species plays a key role in signaling from cAMP to p38-MAPKs. Inhibition of the p38-MAPK pathway with SB203580 partially but significantly, attenuates cAMP-and TSH-induced expression of the sodium iodide symporter in FRTL-5 cells. These results point to a new signaling pathway for the G s -coupled TSH receptor, involving cAMP, protein kinase A, Rac1, and reactive oxygen species and resulting in the activation of a signaling kinase cascade that includes MAPK kinase 3 or 6, p38-MAPK, and MAPK-activated protein kinase-2.The activation of mitogen-activated protein kinases (MAPKs) 1 is a key event in many cellular processes, including proliferation, differentiation, and apoptosis (1). There are three main classes of MAPK, extracellular signal-regulated protein kinases (ERKs) (2, 3), stress-activated protein kinases, also known as c-Jun NH 2 -terminal protein kinases (JNKs) (4, 5), and p38-MAPKs (6 -11). p38-MAPKs ␣, ␤, ␥, and ␦ are activated by the dual phosphorylation of threonine and tyrosine residues within the tripeptide motif TGY (12) by MAP kinase kinases termed MKK3 and MKK6, themselves activated by phosphorylation (13). The p38-MAPKs phosphorylate other protein kinases, such as MAPKAP kinase-2/3 (14, 15) and transcription factors, such as ATF2, Elk1, MEF2,. The pyridinylimidazole compounds SB203580 and SB202190 are very specific inhibitors of p38␣-and p38␤-MAPKs (21, 22). The Rho family GTPases Rac1 and CDC42 and the STE20-related protein kinases PAK1, PAK3, and germinal center kinase ...