The coding sequence for the murine cytomegalovirus (MCMV) nonstructural phosphoprotein pp50 has previously been mapped to the EcoRI-L fragment of the viral genome. Nucleotide sequencing of both genomic DNA and cDNA clones revealed an open reading frame of 411 amino acids that is homologous to both the human cytomegalovirus (HCMV) UL44 (ICP36) and the human herpesvirus type 6 (HHV-6) p41 open reading frames. Four of the five conserved cysteine residues are located within the highly conserved N-termini of the three proteins. Similar to the HCMV and HHV-6 proteins, pp50 is predicted to have a hydrophilic C-terminus which contains a putative nuclear targeting sequence. Immunofluorescence experiments with monoclonal antibodies specific for pp50 demonstrated that recombinant pp50 expressed transiently in COS-1 cells or during infection by recombinant Vaccinia virus was localized to the nucleus. Similarly, during MCMV infection of Balb/3T3 fibroblasts, pp50 was present in the nuclei of infected cells between 8 and 24 hr postinfection, suggesting that the protein contains a functional nuclear targeting sequence. In addition, we have demonstrated that pp50 is a DNA-binding protein with affinities for both single-stranded and double-stranded DNA. Finally, results from our analysis by nonreducing and native gel electrophoresis, as well as fractionation by sucrose gradients suggested that pp50 expressed in the absence of viral infection can form disulfide-linked homopolymers. Such homopolymers were also present in MCMV-infected cells.