Gene transfer into a panel of non-small cell lung cancer (NSCLC) cells by adenoviral (Ad) and retroviral (RV) vectors was studied. Indexed to multiplicity of infection (MOI), Ad vectors transduce squamous, adenosquamous, and malignant mesothelioma cells with greater efficiency than large cells or adenocarcinoma cells. Transduction-sensitive cells bind the Ad vector with specificity for the Ad fiber knob, and internalize vector efficiently. Transduction-refractory cells bind and internalize vector by less efficient pathways. Like Ad vectors, there is heterogeneity in RV transduction efficiencies of different NSCLC subtypes. With respect to the most common cell type metastatic to the pleural space (adenocarcinoma), amphotropic retroviral vectors transduce cells of this subtype more efficiently (at a lower MOI) than Ad. RV transduction is not solely dependent on cellular replication, and both permissive and refractory cell lines express the mRNA for the amphotropic RV receptor. These observations suggest that neither Ad nor RV vectors will suffice a priori as the optimal gene transfer vehicle, and successful gene therapy of lung cancer may require tumor-specific or patient-specific vectors.