Puumala virus is the causative agent of nephropathia epidemica (NE), a hantavirus infection which occurs widely in northern and central Europe and is generally diagnosed by the indirect immunofluorescence (IF) method. We have now expressed the Puumala virus Sotkamo strain nucleocapsid (N) protein-coding S genome segment as a beta-galactosidase fusion protein in Escherichia coli by using the pEX2 expression vector. The recombinant protein was purified by cutting the protein band from an agarose gel, melting the agarose, and removing the protein by freezing, incubation on ice, and centrifugation. The recovery was about 1 to 5 mg/200 ml of bacterial suspension, sufficient for coating 100 to 500 enzyme immunoassay microtiter plates. In a study of 312 IF-positive and 233 IF-negative serum samples from NE patients, the recombinant-N-protein enzyme immunoassay detected immunoglobulin G antibodies to Puumala virus with 97.8% sensitivity and 98.5% specificity compared with the IF test results. In addition, an immunoglobulin G avidity enzyme immunoassay was developed and used successfully to diagnose acute NE from a single serum sample. The results demonstrate that the bioengineered antigen is suitable for use in routine diagnostic assays for Puumala virus immunity and recent infection.