Adeno-associated virus-2 (AAV) can integrate in a site-specific manner to human chromosome 19 and is currently in phase I clinical trials for cystic fibrosis (CF) at Johns Hopkins Hospital. The goal of this study was to determine the fate of recombinant AAV containing the CFTR cDNA (AAV-CFTR) in an immortalized pseudotetraploid CF bronchial epithelial cell line (IB3-1) established from a patient with CF. Fluorescence in situ hybridization (FISH) and Southern blotting of DNA from IB3-1 cells infected with wild-type (wt) or recombinant AAV-CFTR were performed. CFRH2, an IB3-1 cell line with an estimated 15-20 integrated copies of CFTR cDNA, was used to test FISH sensitivity. All metaphase spreads had integrated copies: a single site in 36 of 56 (64.3%) and two sites within the same metaphase spread in 20 of 56 (35.7%). 3-CF-8, an IB3-1 cell line with integration of a partial CFTR cDNA (3.9 kb) was also analyzed by FISH. Integration was observed in 56 of 157 (35.7%) metaphase spreads examined. IB3-1 cells infected with wild-type AAV showed integration in 51 of 86 (59%) metaphase spreads examined. Of 51 integrations, 48 (94%) were to chromosome 19. Examination of 67 metaphase chromosome spreads of IB3-1 cells infected with AAV-CFTR vector (Azero) identified four integrations (6%) to different chromosomes. No integration was to chromosome 19 which differs significantly (P < 0.0001) from wild-type AAV. We then analyzed the A35 cell line, a clone of Azero selected for stable CFTR expression. Genomic DNA from A35 cells did not show a single site of integration; however episomal AAV-CFTR sequences were abundant in the low molecular weight DNA fraction. Examination of 68 metaphase chromosome preparations identified eight distinct integrations, none to chromosome 19. These studies show that FISH is sensitive for the detection of a partial CFTR cDNA integration. Wild-type AAV integrates in a predominantly site-specific fashion. Recombinant AAV-CFTR integrates at low frequency in a nonspecific manner and persists in episomal form in this epithelial cell line.