Normal cells irreversibly stop dividing after being exposed to a variety of stresses. This state, called cellular senescence, has recently been demonstrated to act as a tumor-suppressing mechanism in vivo. A common set of features are exhibited by senescent cells, but the molecular mechanism leading to the state is poorly understood. It has been shown that p38, a stress-induced mitogen-activated protein kinase (MAPK), plays a pivotal role in inducing cellular senescence in diverse settings. To better understand the senescence-inducing pathway, microarray analyses of normal human fibroblasts that ectopically activated p38 were performed. It was found that five genes encoding ESE-3, inhibin betaA, RGS5, SSAT and DIO2 were up-regulated in senescent cells induced by RasV12, H(2)O(2) and telomere shortening, but not in quiescent or actively growing cells, suggesting that these genes serve as molecular markers for various types of cellular senescence. The ectopic expression of ESE-3 resulted in retarded growth, up-regulation of p16(INK4a) but not of p21, and increased levels of SA-beta-gal activity. In contrast, RGS5, SSAT and the constitutive active form of the inhibin betaA receptor gene did not induce such senescence phenotypes when ectopically expressed. ESE-3 expression increased the activity of the p16(INK4a) promoter in a reporter assay, and recombinant ESE-3 protein bound to the Ets-binding sequences present in the promoter. These results suggest that ESE-3 plays a role in the induction of cellular senescence as a downstream molecule of p38.