A new blocking enzyme-linked immunosorbent assay (ELISA) for detection of mumps virus (MuV) specific antibodies in large numbers of human serum samples was developed. The blocking ELISA is based on the reaction of MuV-specific, conjugated monoclonal antibodies (mAbs) with immobilized virus antigen, that has previously been incubated with a two-fold dilution of human serum. Mouse hybridomas that produce antibodies against MuV proteins were generated. They could be divided into 4 groups according to their hemagglutination inhibiting- and virus neutralizing capacities and their reaction in the blocking ELISA with MuV strain Enders. Ascites material from 22 mAbs derived from the 4 groups was further characterized with the MuV strains Enders and Jeryl Lynn. When mAbs from different groups were mixed in the blocking ELISA, an additional increase in absorbance could be observed. A mixture of 2 MuV neutralizing mAbs that were directed against HN and F protein, was used to assay 3 consecutive pre-, early post- and late postvaccination serum samples of 138 children, vaccinated at the age of 1.5 yr. A correlation of 94% was found between the blocking ELISA and the normal indirect ELISA, and of 98% between the blocking ELISA and the neutralization enzyme immunoassay (N50-EIA). The specificity and rapidity of the blocking ELISA makes it suitable for routine use in the determination of MuV neutralizing antibodies in large quantities of serum samples.