Gene transfer to the lung could provide important new treatments for chronic and acquired lung diseases such as cystic fibrosis, alpha1-antitrypsin deficiency, emphysema, and cancer. DNA-mediated gene transfer to the lung has been previously demonstrated, but anticipated effectiveness has been limited by low gene transfer efficiencies and by transient expression of the transgene. Here, we combine plasmid-based gene transfer with the integrating capacity of the nonviral Sleeping Beauty (SB) transposon vector system to mediate gene insertion and long-term gene expression in mouse lung. We observed transgene expression after 24 h in lungs of all animals injected with the luciferase transposon (pT/L), but expression for up to 3 months required codelivery of a plasmid encoding the Sleeping Beauty transposase. We also observed long-term expression in pT/L-injected animals transgenic for SB transposase. Transgene expression was localized to the alveolar region of the lung, with transfection including mainly type II pneumocytes. We used a linker-mediated PCR technique to recover transposon flanking sequences, demonstrating transposition of pT/L into mouse chromosomal DNA of the lung.