Recombinant adeno-associated virus vectors (rAAV) have been successfully used for long-term gene expression in animal models and in patients. However, while the therapeutic potential of rAAV appears promising, safety issues, including contaminants found in vector stocks, must be further evaluated. We previously reported that a cis-acting replication element present within the AAV-2 p5 promoter was responsible for the encapsidation of rep-cap sequences observed during rAAV production. In that study, we also noticed that plasmid-derived prokaryotic sequences (such as the ampicillin resistance gene) could be found packaged into AAV capsids. In this report, first we confirmed and extended the latter observation by analyzing rAAV stocks produced using different procedures. Second, we demonstrated that these plasmid-derived sequences were transferred and persisted in vivo after rAAV injection into different tissues. Third, our data showed that at least some of these packaged plasmid molecules were linked to the AAV ITRs and were present in vivo in a form that could be rescued through bacterial transformation. This study highlights the need for more stringent characterization of rAAV stocks and provides useful information on the development of rAAV production methods that are able to circumvent or limit the generation of such undesirable particles.