Upon transfection of Rat-2-TK- cells with plasmid pES, containing the cloned 7.0-kilobase (kb) EcoRI-SalI fragment (0.063 to 0.089 map units) of the human cytomegalovirus genome, major immediate-early antigen expression was obtained in 1 to 2% of the nuclei of the transfected cells, as determined by immunofluorescence with the E3 monoclonal antibody. Cotransfection of pES with the cloned herpes simplex virus type 1 thymidine kinase gene resulted in the establishment of a hypoxanthine-aminopterin-thymidine-resistant cell line which expressed a major immediate-early antigen in approximately 1% of the cells at early passages, with expression gradually declining to less than 0.1% upon subculturing. Southern blot analysis of DNA extracted from this cell line revealed the presence of multiple integration events of pES DNA sequences into cellular DNA, including a head-to-tail tandem array of approximately 10 copies of pES. The integration pattern was stable for at least 80 passages. Metaphase chromosomes prepared from this cell line showed, upon in situ hybridization, a strong hybridization signal in both sister chromatids of a large submetacentric chromosome which is considered to have harbored the tandemly integrated pES molecules. Whereas in most cells of the population, immediate-early expression seemed to be repressed, this repression could be overcome by protein synthesis inhibition, resulting in a massive induction of human-cytomegalovirus-specific transcripts of 2.1 and 1.9 kb and a minor species of 2.9 kb. After release from protein synthesis inhibition, approximately 20% of the cells showed nuclear fluorescence when the E3 monoclonal antibody was used.