The inverted terminal repeat (ITR) is the only cis element of AAV genome essential for rAAV rescue, replication and packaging. It is prone to mutation or loss when it is latent in host cell or in plasmid. Plasmids with different ITR types were cloned to compare the influence of ITR types on the AAV packaging and infectivity. The vector plasmids were transformed the competent SURE cells to get different colonies. The ITR types of plasmids were screened by digestion with SmaⅠ. AAV vector plasmid pScGFPud has two ITRs at both ends of AAV genome and plasmid pScGFPu has only one ITR at upstream end of AAV genome. When the two plasmids were co-transfected 293 cells to prepare rAAVs, 1.08×10¹³ viral particles (AAV1-GFPud) were produced from 20-dishes of 293 cells cotransfected with plasmid pScGFPud, 4.28×10¹² viral particles (AAV1-GFPu) were produced from 20-dishes of 293 cells cotransfected with plasmid pScGFPu. Virus AAV1-GFPud infected 293, HeLa and NCI H446 cells more efficiently than did virus AAV1-GFPu. This suggests that defect ITRs in AAV genome is deleterious to AAV packaging and AAV infectivity and vector with complete ITRs is favorable to the yield and activity of rAAV.