Development of gene transfer vectors with regulated, lung-specific expression will be a useful tool for studying lung biology and developing gene therapies. In this study we constructed a series of lentiviral vectors with regulatory elements predicted to produce lung-specific transgene expression: the surfactant protein C promoter (SPC) for alveolar epithelial type II cell (AECII) expression, the Clara cell 10-kD protein (CC10) for Clara cell expression in the airway, and the Jaagskiete sheep retrovirus (JSRV) promoter for expression in both cell types. Transgene expression from the SPC and CC10 vectors was restricted to AECII and Clara cell lines, respectively, while expression from the JSRV vector was observed in multiple respiratory and nonrespiratory cell types. After intratracheal delivery of lentivector supernatant to mice, transgene expression was observed in AECII from the SPC lentivector, and in Clara cells from the CC10-promoted lentivector. Transgene expression was not detected in nonrespiratory tissues after intravenous delivery of CC10 and SPC lentiviral vectors to murine recipients. In summary, incorporation of genomic regulatory elements from the SPC and CC10 genes resulted in respiratory specific transgene expression in vitro and in vivo. These vectors will provide a useful tool for the study of lung biology and the development of gene therapies for lung disorders.