Four classes of monoclonal antibody-producing cell lines have been obtained that detect human cytomegalovirus virion structural proteins. These antibodies react with (1) a major outer membrane virion glycoprotein(s) gp58-gp130, whose molecular weight varies between strains of cytomegalovirus, (2) a phosphoprotein, pp71, localized inside the virion membrane, (3) a phosphorylated nucleocapsid protein, pp155, and (4) a virion-associated phosphoprotein, pp29. Polyclonal immune human sera react with a large number of virion proteins including those detected by these monoclonal antibodies. These monoclonal antibodies were employed in a radioimmune assay to detect low levels (6 X 10(3) PFU/ml) of human cytomegalovirus in solution and human urine. These antibodies were also employed in a fluorescent antibody format to identify cytomegalovirus-infected cells obtained from human urine and nasopharyngeal aspirates. These reagents provide useful tools for studying the molecular biology of virus replication, for diagnosing cytomegalovirus infections, and for studying virus latency and activation.