We have identified an abundant 50K phosphoprotein (pp50) in MCMV-infected 3T3-L1 cells and shown by immunofluorescence microscopy and surface-iodination experiments that pp50 is localized to the plasma membrane of the infected cell. Furthermore, the kinetics of its synthesis suggests that it belongs to the early-late class of herpesvirus proteins. Using monoclonal antibodies specific for pp50 to screen a lambda ZAP II expression library constructed from poly(A)+ mRNA of MCMV-infected cells, we have isolated a cDNA clone that synthesizes a truncated form of pp50 as a beta-galactosidase fusion protein. This allowed us to localize the partial pp50 transcript to a region between map coordinates 0.228 and 0.260 of the MCMV genome (Smith strain, Vancouver). Finally, we demonstrated that the MAb 5H10.21A recognizes an antigenic determinant that is conserved between pp50 and a 50K human cytomegalovirus (HCMV) nonstructural protein.