Lung diseases such as cystic fibrosis (CF) might be treated by gene therapy using viral vectors delivered to the airway. One potential vector is the defective human parvovirus, adeno-associated virus (AAV). We examined the AAV p5 transcription promoter for gene expression in immortalized cell lines derived from the airway (IB3-1) or pancreas (CFPAC-1) of CF patients. AAV vectors expressing the prokaryotic genes cat (pAAVp5cat) or neo (pAAVp5neo) from the p5 promoter were evaluated after introduction into IB3-1 or CFPAC-1 cells by lipofection. In transient assays in both cell lines, the cat gene was expressed 5- to 10-fold more efficiently from the p5 promoter than from a simian virus 40 early gene promoter (pSVcat). IB3-1 cells were transformed stably to geneticin resistance by pAAVp5neo at a 5-fold higher efficiency than by an SVneo vector. The AAV inverted terminal repeat (ITR) region immediately upstream of the p5 promoter appears to have an enhancer effect and the promoter also contains a CREB site which confers a response to forskolin. In IB3-1 cells, expression of the cat gene from a p5 promoter was decreased about 5-fold by deletion of both the upstream ITR and the CREB site. The AAVp5neo vector was also packaged into AAV particles and used to infect IB3-1 cells as a transducing virus. Under these conditions, 60 to 70% of the cells could be stably transformed to geneticin resistance. Thus, AAV transducing vectors appear to be a highly efficient delivery system for stable integration and expression of genes in cultured airway epithelial cells.