A gene of rat cytomegalovirus (RCMV), designated R32, has been identified that encodes a homologue of the human cytomegalovirus (HCMV) pp150 (ppUL32) major tegument phosphoprotein. The R32 ORF has the capacity to encode a 667 amino acid polypeptide (pR32) with a calculated molecular mass of 73 kDa. The predicted amino acid sequence of pR32 shows similarity to that of polypeptides predicted to be encoded by the HCMV UL32, murine cytomegalovirus M32 and human herpesvirus types 6 and 7 U11 genes. The R32 gene is transcribed as a 2.5 kb mRNA during the late phase of RCMV infection in rat embryo fibroblasts in vitro. To study expression of the pR32 protein in vitro and in vivo, a rabbit polyclonal antiserum was raised against a recombinant protein that comprised amino acids 252-522 of pR32. By using this antiserum, pR32 could be detected predominantly in the cytoplasm of RCMV-infected fibroblasts at 24 and 48 h post-infection in vitro. The pR32 protein was also detected within virions isolated from the culture medium of RCMV-infected cells. Expression of pR32 in vivo was observed within the cytoplasm of salivary gland epithelial cells of RCMV-infected rats. In addition, recombinant pR32 was found to react with sera from rats that were previously infected with RCMV, whereas reactivity was not seen with sera from mock-infected rats. Together, these findings indicate that RCMV pR32 represents the homologue of HCMV ppUL32, both in primary structure and in function.