There is evidence that reactive oxygen species (ROS) are important mediators of tumor promotion and progression. The molecular mechanisms involved in ROS-mediated signaling, however, are unclear at present. Using ionizing radiation and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as model physical and chemical carcinogens, we have malignantly progressed 308 cells, a papilloma-producing mouse keratinocyte cell line, and investigated the molecular alterations in the progressed phenotypes. In this study, we demonstrate that both MNNG and radiation-progressed malignant variants showed elevated ROS levels that contributed to their proliferative capacity in vitro as well as in vivo. We found increased Erk-1/2 and p38 MAP kinase activities to be important components of ROS-mediated signaling. The pro-oxidant state also contributed to constitutive elevation of AP-1, NFkappaB and cAMP response element transactivation in the malignant phenotype. Our data provide evidence for a functional role of elevated ROS levels in tumor progression and implicate Erk-1/2 and p38 MAP kinase activation in the malignant progression of mouse keratinocytes.