A new immunoglobulin M (IgM) antibody capture enzyme immunoassay with peroxidase-labeled mumps antigen (dMACEIA) is described, and its suitability for practical diagnosis of acute mumps infection is evaluated. All 54 patients with proven mumps infection that were tested showed mumps-specific IgM antibodies. On the other hand, no specific IgM antibodies were present in 16 cases of suspected mumps that could not be confirmed by classical complement fixation serology, and IgM mumps virus antibodies could be detected neither in the sera of 100 healthy individuals nor in those of 16 patients positive for rheumatoid factor. In all, 22 children with acute respiratory illness caused by parainfluenza virus and 44 patients with infections due to other viruses showed no IgM response in mumps dMACEIA. The particular characteristic in which complement fixation antibodies against mumps nucleocapsids appear before and disappear earlier than antibodies to the enveloped mumps virus could not be demonstrated in the dMACEIA. In an extensive epidemic of mumps virus infection, the dMACEIA gave a clear diagnosis of mumps infection in 200 out of 371 suspected cases. By day 2 of the illness, 71% of the patients had detectable IgM, and by day 3, all of them had detectable IgM. In 99% of the cases, dMACEIA gave a positive result in the first available serum specimens, most of which were negative for complement fixation antibodies. A positive but only moderate correlation was thus observed between the two serological procedures. IgM antibodies persisted for at least 6 weeks. The dMACEIA, performed in 3 h, offers a reliable, simple, and rapid alternative to routine methods for detection of acute mumps infection.