Pseudorabies virus (PRV) is an alphaherpesvirus, and its gene organization and regulation are similar to the well-characterized human herpes simplex virus (HSV). The PRV early protein UL54 consists of 363 amino acids with homology to the HSV ICP27 immediate-early protein. Previously, we have demonstrated the nuclear accumulation and poly(G) RNA-binding activity of UL54 protein. In the present study, we have identified further the functional regions within UL54 conferring for nuclear localization and RNA-binding activity. Several recombinant expression plasmids containing various coding regions of UL54 gene were constructed for producing a series of C-terminally truncated or internally deleted forms of UL54 mutants in Escherichia coli or porcine kidney (PK-15) cells. RNA-binding activity of E. coli-expressed UL54 mutants was characterized by the binding ability to poly(G) RNA homopolymer in dot blot hybridization assay and the results have shown that the N-terminal 83 residues were responsible for RNA-binding, and the region of residues 35-82 containing an RGG box was necessary for its function. Furthermore, the region responsible for nuclear localization was investigated by transient expression of various deletion mutants in PK-15 cells followed by detection of their subcellular distribution. The results showed that C-terminal deletion beyond the amino acid residue 83 or internal deletion containing the RGG box sequence could restrict UL54 mutants in the cytoplasm. The ability of the N-terminal 83 residues to target the green fluorescence protein to the nucleus confirmed further its role as a functional nuclear localization signal (NLS). The utmost N-terminal 83 residues portion of UL54 contains two important functional domains, NLS and RNA-binding, and thus it would play an indispensable role in UL54 regulatory function.