The proliferation of human non-small-cell-lung-cancer (NSCLC) cells is regulated by the epidermal-growth-factorreceptor (EGFR)-mediated autocrine loop that interacts with transforming growth factor-โฃ (TGF-โฃ) of autocrine or paracrine origin. We have shown that EGFR expression is elevated in the brain metastatic variant of human NSCLC cells H226Br, which thereby acquire their increased sensitivity toward exogenous TGF-โฃ. To determine detailed cellphenotype changes as a result of EGFR down-regulation, H226Br cells were transfected with a human EGFR-cDNA construct encompassing an N-terminal fragment (1.8 kb) in anti-sense orientation downstream of the cytomegalovirus (CMV) promoter. The EGFR transcript expressed in the 3ะ-5ะ direction is expected to neutralize EGFR mRNA and to reduce protein expression correspondingly. The established cell lines resistant to G418 were shown integrated with the transfected construct and their proliferation rates reduced as compared with the parental cells and with those transfected with vector alone. Down-regulated EGFR expression in cells with the anti-sense construct can be confirmed by Scatchard analysis and suppressed EGFR kinase activity. The restrainedgrowth phenotype is also demonstrated in the prolonged G 2 -M phase during the cell cycle, and correlated with impairment of cell proliferation. This finding suggests that EGFR over-expression is critical in maintaining the malignant phenotype of NSCLC cells, thereby providing a valuable biomarker and potential target prevention for lung-cancer-cell proliferation.