Mitogen-activated protein (MAP) kinases are important mediators of the cellular stress response. Here, we investigated the relationship between activation of the MAP kinase p38 and transcription factor NF-kappaB. Different forms of cellular stress were found to preferentially trigger either p38 or NF-kappaB. Arsenite or osmotic stress potently activated p38 but were ineffective in inducing NF-kappaB activation. Tumor necrosis factor-alpha and hydrogen peroxide, in contrast, led to NF-kappaB activation but only modestly stimulated p38. The activation of NF-kappaB was strongly abolished by antioxidants, while the activity of p38 and transcription factor AP-1 were increased. Inhibition of small GTPases including Rac and Cdc42 prevented p38 and AP-1 activation without interfering with NF-kappaB. In addition, inhibition of p38 by a pharmacological inhibitor or a dominant-negative mutant of MAP kinase kinase-6, an activator of the p38 pathway, interfered with NF-kappaB-dependent gene expression but not its DNA binding activity. Our results indicate that activation of p38 and NF-kappaB are mediated by separate pathways, which may converge further downstream in the cell nucleus. Different forms of cellular stress, however, initially trigger distinct signaling cascades involving either oxidative stress or GTPase-coupled pathways.