Adeno-associated virus (AAV) is gaining momentum as a gene therapy vector for human applications. However, there remain impediments to the development of this virus as a vector. One of these is the incomplete understanding of the biology of the virus, including nuclear targeting of the incoming virion during initial infection, as well as assembly of progeny virions from structural components in the nucleus. Toward this end, we have identified four basic regions (BR) on the AAV2 capsid that represent possible nuclear localization sequence (NLS) motifs. Mutagenesis of BR1 (¹²⁰QAKKRVL¹²⁶) and BR2 (¹⁴⁰PGKKRPV¹⁴⁶) had minor effects on viral infectivity (∼4- and ∼10-fold, respectively), whereas BR3 (¹⁶⁶PARKRLN¹⁷²) and BR4 (³⁰⁷RPKRLN³¹²) were found to be essential for infectivity and virion assembly, respectively. Mutagenesis of BR3, which is located in Vp1 and Vp2 capsid proteins, does not interfere with viral production or trafficking of intact AAV capsids to the nuclear periphery but does inhibit transfer of encapsidated DNA into the nucleus. Substitution of the canine parvovirus NLS rescued the BR3 mutant to wild-type (wt) levels, supporting the role of an AAV NLS motif. In addition, rAAV2 containing a mutant form of BR3 in Vp1 and a wt BR3 in Vp2 was found to be infectious, suggesting that the function of BR3 is redundant between Vp1 and Vp2 and that Vp2 may play a role in infectivity. Mutagenesis of BR4 was found to inhibit virion assembly in the nucleus of transfected cells. This affect was not completely due to the inefficient nuclear import of capsid subunits based on Western blot analysis. In fact, aberrant capsid foci were observed in the cytoplasm of transfected cells, compared to the wild type, suggesting a defect in early viral assembly or trafficking. Using three-dimensional structural analysis, the lysine- and arginine-to-asparagine change disrupts hydrogen bonding between these basic residues and adjacent beta strand glutamine residues that may prevent assembly of intact virions. Taken together, these data support that the BR4 domain is essential for virion assembly. Each BR was also found to be conserved in serotypes 1 to 11, suggesting that these regions are significant and function similarly in each serotype. This study establishes the importance of two BR motifs on the AAV2 capsid that are essential for infectivity and virion assembly.