Although alpha-dystrobrevin (DB) is assembled into the dystrophin-associated protein complex, which is central to cytoskeletal organization, it has also been found in the nucleus. Here we delineate the nuclear import pathway responsible for nuclear targeting of alpha-DB for the first time, together with the importance of nuclear alpha-DB in determining nuclear morphology. We map key residues of the nuclear localization signal of alpha-DB within the zinc finger domain (ZZ) using various truncated versions of the protein, and site-directed mutagenesis. Pulldown, immunoprecipitation, and AlphaScreen assays showed that the importin (IMP) alpha2/beta1 heterodimer interacts with high affinity with the ZZ domain of alpha-DB. In vitro nuclear import assays using antibodies to specific importins, as well as in vivo studies using siRNA or a dominant negative importin construct, confirmed the key role of IMPalpha2/beta1 in alpha-DB nuclear translocation. Knockdown of alpha-DB expression perturbed cell cycle progression in C2C12 myoblasts, with decreased accumulation of cells in S phase and, significantly, altered localization of lamins A/C, B1, and B2 with accompanying gross nuclear morphology defects. Because alpha-DB interacts specifically with lamin B1 in vivo and in vitro, nuclear alpha-DB would appear to play a key role in nuclear shape maintenance through association with the nuclear lamina.-Aguilar, A., Wagstaff, K. M., Suarez-Sanchez, R., Zinker, S., Jans, D. A., Cisneros, B. Nuclear localization of the dystrophin-associated protein alpha-dystrobrevin through importin alpha2/beta1 is critical for interaction with the nuclear lamina/maintenance of nuclear integrity.