It has been determined that AAV vector efficiency decreases due to the following mechanisms: ineffective endocytosis, endosomal degradation, inefficient trafficking, and the need to convert from a single-stranded AAV genome to a transcriptional active double-stranded form. Administering rAAV vectors in conjunction with a drug that can enhance vector transduction may overcome some of these cellular barriers encountered during infection as well as help stabilize the genome. Here we discovered that Cidofovir(CDV), a monophosphate nucleotide analogue that competitively inhibits the incorporation of deoxycytidine triphosphate into viral DNA via viral DNA polymerase, is able to increase single-stranded rAAV transgene expression in the nucleus by 2 to 9 fold, depending on the cell type and concentration. This increase has been consistently observed in 16095, HelaS3, and Hek293 cells with little toxicity to the cells. However, there is less than a two-fold increase with Cidofovir treatment when using self-complementary AAV vectors(scAAV), suggesting that CDV might enhance conversion of the AAV genome from single to double stranded form. These observations were confirmed using two different serotypes, AAV2 and AAV8, in vitro indicating that the mechanism of enhancement is not serotype dependent. Southern Blot analysis along with real-time PCR demonstrated that Cidofovir treatment in HelaS3 cells increases viral DNA accumulation 24 hours post-infection compared to PBS treated cells. Cells pretreated with CDV then infected with rAAV revealed no difference in the amount of vector present between 0 and 2 hrs post infection, suggesting CDV does not enhance viral entry. However, cytoplasmic and nuclear fractions of HelaS3 cells pretreated with CDV or PBS demonstrated that CDV pretreatment enhances viral accumulation of rAAV vectors in both the cytoplasm and nucleus 24 hours post-infection.In vivo, Hemophilia A BALB/c(BALB/c-HA) mice were treated with CDV prior to infection with a rAAV containing the fVIII Human Light chain(LC) gene. It showed that CDV treatment enhanced the amount of LC antigen when compared to control group. Approximately two fold increase was observed up to fourteen weeks post-injection. These results demonstrate that CDV treatment can enhance and sustain rAAV levels in vivo.CDV has already been approved by the Food and Drug Administration (FDA) for the treatment of cytomegalovirus retinitis in patients with acquired immunodeficiency syndrome (AIDS). The effects of CDV on small DNA viruses that lack their own viral DNA polymerase, like AAV, have not been documented. The use of drugs like Cidofovir can greatly contribute to the understanding of rAAV trafficking and eventually lead the development of novel strategies to increase overall efficiency of AAV transduction.