Human papillomaviruses (HPVs) such as types 6 and 11 can establish lifelong infections in airway epithelial cells in patients, and long-term infection can lead to pulmonary involvement and death. The mechanisms underlying this persistence depend on both the transcriptional activity of the viral enhancers and promoters and the ability of this virus to maintain its double-stranded circular DNA genome in infected tissues. We investigated the transcription and replication properties of HPV sequence elements and protein products in a human airway cell line. We showed that incorporation of the upstream regulatory region and cotransfection with expression vectors of two virus-encoded proteins, E1 and E2, conferred approximately 5,000-fold stimulation of reporter gene expression. Transient plasmid replication in transfected human airway cells and lungs of FVB/N-C57BL/6 mice was demonstrated by a modified transient replication assay. These results have important implications for viral pathogenesis in airway cells and the potential of HPV-based replicons for gene transfer into airway epithelium.