Alcelaphine herpesvirus-1 (AlHV-1) is a γ2 rhadinovirus associated with Malignant Catarrhal Fever (MCF) in cattle. ORF 57 is well conserved among gammaherpesviruses and it has been shown that the ORF 57 gene products of Herpesvirus Saimiri (HVS), Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) play an important role in regulating viral gene expression. The AlHV-1 ORF 57 gene product has not been characterized. In the accompanying paper we have demonstrated that AlHV-1 ORF 57 encodes an immediate early protein that acts as a regulator of gene expression. The ORF 57 gene product has an up-regulatory effect only on another immediate early gene product encoded by ORF 50. Here we show that the ORF 57 gene product is a nuclear protein. When ORF 57 was fused to the gene encoding Enhanced Green Fluorescent Protein (EGFP), the fusion protein exhibited a punctate nuclear distribution that co-localized with the nucleolar phosphoprotein C23. The nuclear localisation signal of ORF 57 gene product was located at the N-terminus. The ORF 57 gene product travels from nucleus to the cytoplasm, where it accumulates during Actinomycin D treatment. The domain involved in nuclear shuttling was also localised at the N-terminal region of the protein. Thus in common with homologues in other herpesviruses the AlHV-1 ORF 57 gene product is a nuclear cytoplasmic shuttling protein which may play a role in export of viral mRNAs from the nucleus of infected cells.