Tumor cell motility is one of the rate-limiting steps of invasion, which defines progression toward a more malignant phenotype. Elevated expression of epidermal growth factor receptor (EGFR) in many cancers is associated with progression of superficial to invasive forms of the disease. The naturally occuring type III mutant epidermal growth factor receptor (EGFRvIII) is a tumor-specific, ligand-independent, constitutively active variant of the epidermal growth factor receptor. EGFRvIII is expressed frequently by a number of human solid tumours including those of the lung, breast, prostate, brain and ovary. Our study was designed to investigate the effect of EGFRvIII expression on cell motility and compare it to that of ligand-activated EGFR using transfected fibroblasts. We show here using time-lapse video recording that expression of EGFRvIII greatly enhances the motility of fibroblasts independently of ligand stimulation. In addition, expression of EGFRvIII caused a marked increase in the number of cellular protrusions (lamellipodia) and a reduction in the number of stress fibers and focal adhesions. The EGFR tyrosine kinase inhibitor, AG1478, and the MEK inhibitor, U0126, blocked these cellular effects of EGFRvIII. Two cell lines expressing different levels of EGFR were used for comparison. The low-expressing cell line responded to EGF treatment by increasing motility in a manner very similar to the motility induced by EGFRvIII. In contrast, the high-expressing cell line responded to EGF by detachment from the extracellular matrix and decreased motility. Cellular detachment was correlated to a high phosphorylation of PLC-γ, whereas increased motility was correlated to a high level of ERK phosphorylation. Overall these results indicate that tumor-associated EGFR mutations might be critical for tumor cell motility, invasion and thus progression of disease. © 2003 Wiley-Liss, Inc.