The Fas receptor and ligand initiate an apoptotic pathway. Alterations in this pathway within tumor cells can result in escape from apoptosis and immune surveillance. We evaluated Fas protein expression in 42 primary pulmonary adenocarcinomas, and Fas expression and function in the lung adenocarcinoma cell lines A549 and A427. Immunohistochemical analysis demonstrated Fas protein expression in 47.6% of the tumors; however, Fas-positive tumors demonstrated cytoplasmic staining without cell surface expression. Northern blot analysis indicated that levels of Fas mRNA were similar in Fas protein-positive tumors to levels in normal lung tissue, but were reduced in Fas protein-negative tumors. Soluble form Fas was not detected in the majority of these tumors either by RT-PCR or Western blot analysis. Cell surface Fas protein expression was minimal in A549 and A427 cell lines as determined by flow cytometry. Both cell lines demonstrated Fas mRNA expression by Northern blot analysis and abundant protein expression by Western blot analysis. Transfection of the Fas cDNA derived from A549 cells induced surface Fas protein in COS cells; however, stable transfection of a native Fas cDNA into A549 cells failed to induce surface Fas protein expression. Parental A549 cells and A549 cells transfected with a Fas expression vector were resistant to Fas-mediated apoptosis. Transgenic expression of a FLAG-tagged Fas cDNA in A549 cells, with visualization of the Fas-FLAG protein using confocal microscopy, demonstrated that the Fas-FLAG protein was retained within cytoplasmic portions of the cell and was not translocated to the cell surface. These findings suggest that the Fas protein is reduced or not present on the cell surface in the primary lung tumors and is sequestered within A549 tumorigenic lung cells, and these alterations directly affect the cells resistance to Fas-mediated apoptosis.