Early nuclear antigen (CMNA) induced by human cytomegalovirus (HCMV) was extracted from infected human embryonic fibroblast cells and purified by ds DNA Sephadex chromatography. The purified antigen was added to acid-fixed preparations from human embryonic fibroblasts and these in vitro converted nuclei were exposed to human sera to estimate the antibodies to CMNA by anti-complement immunofluorescence staining. Serial serum samples were obtained from eight patients suffering from acute HCMV infection (mononucleosis), from nine patients with Epstein-Barr virus (EBV) mononucleosis, and from 20 healthy persons who had been shown to possess antibodies to HCMV late antigens. Acute HCMV infection is characterized by the presence of anti-CMNA antibodies at high titer (1:16 - 1:32) together with the elevated level of IgG antibodies to anti-HCMV-late antigens. During convalescence the anti-CMNA titer decreased to a lower level which was maintained for long period. The anti-CMNA antibodies were also regularly detected in sera of persons possessing antibodies to HCMV late antigens but without any sign of acute HCMV infection. It is concluded that antibodies to CMNA are not transitory and their presence even at high titer (1:16 - 1:32) in a serum sample cannot be taken as a presumptive evidence of acute virus infection. This method seems to be valuable for measuring antibodies to nuclear DnA-binding antigens induced by other viruses.