Epidermal growth factor receptor (EGF) variant type III (EGFRvIII) is a constitutively active, naturally occurring mutation of the EGF receptor that is found in many types of human tumors. When overexpressed in NIH3T3 fibroblasts, EGFRvIII induces transformation by enhancing cell growth and reducing apoptosis. Analysis of downstream signaling pathways has revealed that extracellular signal-regulated kinase activity is down-regulated, raising doubt as to the significance of this pathway in promoting transformation. We investigated whether the c-Jun N-terminal kinase (JNK) pathway was affected by EGFRvIII. NIH3T3 cells expressing EGFRvIII exhibited a high basal level of JNK activity, which was not present in cells overexpressing the normal EGF receptor. Treatment of cells overexpressing EGFRvIII with inhibitors of the EGF receptor or phosphatidylinositol 3-kinase resulted in the down-regulation of JNK activity. Furthermore, the down-regulation of JNK activity was associated with a loss of properties related to transformation, and there was no evidence for JNK activity in the promotion of apoptosis in these cells. These findings implicate constitutive activation of the JNK pathway in transformation by EGFRvIII.