Adeno-associated virus type 2 (AAV2)-based vectors are capable of stable expression in the airway epithelium and may be useful for gene therapy for human diseases, such as cystic fibrosis. Certain virus vectors, such as retroviruses, require active cell division for integration and expression, but this has not been formally evaluated in the case of AAV2. The cystic fibrosis bronchial epithelial cell line, IB3-1, which can be transduced by AAV2 vectors, was shown to undergo a decrease in DNA synthesis to undetectable levels when grown to confluence. Cultures in which < 0.1% of cells were dividing could still be efficiently transduced with AAV-lacZ or AAV-neo vectors, with a linear dose response, up to 91% with a multiplicity of 3,000 vector particles per cell. The fate of vector DNA in nondividing target cells was investigated by Southern blotting of both low molecular weight, nonintegrated DNA and high molecular weight, genomic DNA fractions. Detectable levels of vector DNA were only seen in the nonintegrated state. These results indicate that AAV2-based vectors, unlike retrovirus vectors, do not require active cell division or integration for expression to occur and thus possess a unique profile of biologic properties.