An enzyme-linked immunosorbent assay (ELISA) was developed to demonstrate cytomegalovirus (CMV) antibodies and antigen. A nuclear antigen from CMV-infected cells was used for detection of IgG and IgM antibodies. Significant rises of CMV-IgG and significant levels of CMV-IgM were found in sera from patients with acute CMV only, and not with other herpesvirus infections. CMV antigens were demonstrated in cells and in culture medium by a sandwich or by an inhibition ELISA technique. In the sandwich technique, the plates were coated with monkey anti-CMV IgG and rabbit anti-CMV IgG was used as the second antibody. Both early and late CMV antigens were identified with this method. Treatment of CMV containing samples using freeze--thawing or detergent reduced the infectivity, but the antigenicity was not affected. There was no cross-reactivity with herpes simplex or varicella-zoster virus.