In view of developing an enzyme-linked immunosorbent assay (ELISA) for the determination of IgG antibody to human cytomegalovirus, a rapid microneutralization (Nt) assay was used to test five positive standard sera containing increasing amounts of specific antibody and a negative standard serum. The standard serum containing the minimal amount of detectable Nt antibody was selected as a cut-off standard for the ELISA test. Following preliminary testing on previously characterized sera which gave expected results, the ELISA assay was tested in the field on 992 sera from blood donors. In parallel, sera were tested by Nt and complement fixation (CF). ELISA detected 82 negative and 910 positive sera. Nt gave concordant result except for two ELISA-negative sera, which showed Nt antibody titers of 1:10. The absorbance value of these two sera was just below that of the cut-off. Thus, for ELISA, the sensitivity was 99.8% (910/912) and specificity 100% (80/80). CF gave results concordant with ELISA and Nt, except for 23 sera (2 ELISA- and Nt-negative, and 21 ELISA- and Nt-positive) showing anticomplementary activity. Quantitation of specific ELISA antibody was achieved by interpolation from a calibation curve. Nt appears to be the reference test to establish the ELISA cut-off.