Cellular senescence, initially observed during subculturing of normal diploid fibroblasts, can also be induced by chronic exposure to cellular stress, such as UV light, oxidative stress, or DNA damaging agents. Here we demonstrate that stable expression of an activated form of MKK6 (MKK6EE), a direct activator of the stress-induced p38(HOG) mitogen-activated protein kinase pathway, is sufficient for inducing features of senescence including a flattened, vacuolated, and irregular morphology, staining for acidic beta-galactosidase, and accumulation of age-associated pigments. Consistent with the senescent phenotype, p38(HOG) activation induces a G(1) cell cycle arrest, which is permanent and irreversible after 4 days. MKK6EE also induces biochemical features of senescence in a p38-dependent manner, including enhanced expression of p21(CIP), a cyclin-dependent kinase inhibitor. Microarray analysis of MKK6EE cells showed a pattern of gene expression noted previously in Werner Syndrome and senescent fibroblasts. These results define p38(HOG) as an intracellular pathway that activates a senescence checkpoint in tumor cells and may play a role in Ras- or stress-induced senescence.