The L6 region of bovine adenovirus (BAdV)-3 encodes a spliced protein designated 33K. The 33K specific sera detected five major proteins and three minor proteins in transfected or virus infected cells, which could arise by internal initiation of translation and alternative splicing. The 33K protein is predominantly localized to the nucleus of BAdV-3 infected cells. The 33K nuclear transport utilizes both classical importin-α/-β and importin-β dependent nuclear import pathways and preferentially binds to importin-α5 and transportin-3 receptors, respectively. Analysis of mutant 33K proteins demonstrated that amino acids 201-240 of the conserved C-terminus of 33K containing RS repeat are required for nuclear localization and, binding to both importin-α5 and transportin-3 receptors. Interestingly, the arginine residues of conserved RS repeat are required for binding to transportin-3 receptor but not to importin-α5 receptor. Moreover, mutation of arginines residues of RS repeat proved lethal for production of progeny virus. Our results suggest that arginines of RS repeat are required for efficient nuclear transport of 33K mediated by transportin-3, which appears to be essential for replication and production of infectious virion.