The L2 region of bovine adenovirus-3 (BAdV-3) encodes a genus-specific protein, designated as pV, which is important for the production of progeny viruses. Here, we demonstrate that BAdV-3 pV, expressed as 55 kDa protein, localizes to the nucleus and specifically targets nucleolus of the infected cells. Analysis of deletion mutants of pV suggested that amino acids 81-120, 190-210, and 380-389 act as multiple nuclear localization signals (NLS), which also appear to serve as the binding sites for importin α-3 protein, a member of the importin α/β nuclear import receptor pathway. Moreover, pV amino acids 21-50 and 380-389 appear to act as nucleolar localization signals (NoLs). Interestingly, amino acids 380-389 appear to act both as NLS and as NoLS. The presence of NoLS is essential for the production of infectious progeny virions, as deletion of both NoLs are lethal for the production of infectious BAdV-3. Analysis of mutant BAV.pVd1d3 (isolated in pV completing CRL cells) containing deletion/mutation of both NoLS in non-complementing CRL cells not only revealed the altered intracellular localization of mutant pV but also reduced the expression of some late proteins. However, it does not appear to affect the incorporation of viral proteins, including mutant pV, in BAV.pVd1d3 virions. Further analysis of CsCl purified BAV.pVd1d3 suggested the presence of thermo-labile virions with disrupted capsids, which appear to affect the infectivity of the progeny virions. Our results suggest that pV contains overlapping and non-overlapping NoLS/NLS. Moreover, the presence of both NoLS appear essential for the production of stable and infectious progeny BAV.pVd1d3 virions.