Human parvovirus B19 is the aetiological agent of the common childhood disease erythema infectiosum (fifth disease). The infection is usually benign and self-limiting, but in adults cases of severe arthritis which may persist for years have been reported. Neutralizing antibodies directed against the structural proteins are usually produced shortly after the infection. The immune response against the third major protein, the non-structural protein NS-1, of parvovirus B19 has not been characterized so far. We cloned and expressed the full-length NS-1 protein and fragments thereof in Escherichia coli. The purified recombinant proteins were used to investigate the presence of antibodies to the NS-1 protein in sera from patients with parvovirus B19 infection. Specific antibodies could be detected in sera from patients suffering from severe parvovirus B19-associated arthritis using Western blot analysis and an ELISA. Sera from patients with acute or past infection without complications did not contain detectable levels of immunoglobulin to NS-1. The use of subfragments of the NS-1 protein allowed localization of the antigenic domains in the carboxy-terminal region of the protein.