beta-dystroglycan (beta-DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of beta-DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of beta-DG, characterizing a functional nuclear localization signal (NLS) in the beta-DG cytoplasmic domain, within amino acids 776-782. The NLS either alone or in the context of the whole beta-DG protein was able to target the heterologous GFP protein to the nucleus, with site-directed mutagenesis indicating that amino acids R(779) and K(780) are critical for NLS functionality. The nuclear transport molecules Importin (Imp)alpha and Impbeta bound with high affinity to the NLS of beta-DG and were found to be essential for NLS-dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of beta-DG may result in cytoplasmic retention, with Y(892) playing a key role. beta-DG thus follows a conventional Impalpha/beta-dependent nuclear import pathway, with important implications for its potential function in the nucleus.