Adeno-associated Virus (AAV) has emerged as a promising vector for gene therapy because of its ability to generate high titer recombinant stocks and the potential for site specific integration. However, much of the current knowledge regarding the transduction and integration biology of this virus is based on studies evaluating wild type AAV or recombinant AAV which was unknowingly contaminated with wild type virus. Given the fact that recombinant AAV is replication incompetent, by virtue of deleted viral rep proteins responsible for site specific integration of the wild type virus, the integration process for recombinant AAV may likely be different from its wild type counterpart. To this end, the present study has attempted to elucidate the proviral structure of stably integrated recombinant AAV genomes harboring the alkaline phosphatase reporter gene in 293 and IB3 cell lines. Initial studies attempted to functionally characterize differences in proviral genomes using mobilization assays with assessed both liberated episomal recombinant AAV and infectious virus following transfection with Rep/Cap containing plasmids and/or infection with recombinant adenovirus (Ad). Using Southern and polymerase chain reaction (PCR) analysis, evaluation of genomic DNA from AAV clonal cell lines indicated that head to tail orientations of ITRs were absolutely required for excision of episomal genomes and rescue of infectious recombinant virus. Furthermore, mobilization of proviral DNA could be achieved in the presence of exogenous Rep/Cap without adenovirus, while mobilization of infectious recombinant virus required the addition of both Rep/Cap and Ad. Genomic Southerns suggest that two predominant proviral structures exist for recombinant AAV including head to head and tail to head duplex genomes. A third class of monomer proviral genomes with head to tail oriented ITRs was also observed. No evidence for tail to tail ITR oriented proviral genomes was detected in any of the clonal cell lines. Such findings have begun to lay the foundation for a clearer understanding of the mechanism of recombinant AAV integration and how this process differs from wild type AAV.