A hantavirus infection is followed by a prominent antibody response to the viral nucleocapsid protein. Antibodies from patients infected with one hantavirus cross-react to varying degrees with the nucleocapsid protein of other viruses of the genus. We studied the cross-reactivity in serially obtained blood samples from 17 patients with nephropathia epidemica, a European form of hemorrhagic fever with renal syndrome caused by Puumala virus. Recombinant truncated nucleocapsid protein (aa 1-117) of Puumala virus and four other hantaviruses, Hantaan, Seoul, Dobrava and Sin Nombre viruses, were used as antigens in an indirect ELISA. In most patients, an IgG response to the Puumala virus derived recombinant protein was detected within 2-8 days of onset of disease, remained high for 2-5 months, and declined gradually within 2-3 years. All patients had IgG antibodies cross-reacting with the nucleocapsid protein of Sin Nombre virus. The ratio of the ELISA values obtained with Sin Nombre vs. Puumala virus protein as antigen increased with time after onset of disease. To a lesser extent, cross-reacting IgG antibodies also occurred to Hantaan, Seoul, and Dobrava virus antigens. In the acute phase of the disease, two patients showed IgG antibodies to one or more of these viruses whereas 2-5 months later, 11 of 16 patients had IgG antibodies to all three viruses. IgM and IgA responses to the nucleocapsid protein of Puumala virus were transitory and cross-reactivities were weak. In conclusion, after onset of nephropathia epidemica the IgG response to the Puumala virus nucleocapsid protein was associated with a gradually increasing cross-reactivity to the nucleocapsid protein of heterologous hantavirus. Our findings have implications for the interpretation of serological data, both in the diagnostics of nephropathia epidemica and in seroprevalence studies.