Monoclonal antibodies were raised against a bacterial fusion protein containing amino acids 364 to 623 of the major nonstructural protein, NS-1, of minute virus of mice (MVMp), an autonomous parvovirus. By immunoblot analyses, these antibodies all recognized an 83-kDa protein in MVM-infected mouse fibroblast cells. Indirect immunofluorescence studies showed that five of the six react against a nuclear protein in MVM-infected mouse cells resulting in discrete foci of fluorescence. These foci do not correspond with the nucleoli, the site of MVM DNA replication. The epitopes of the antibodies were mapped using carboxy-terminal deleted bacterial fusion proteins derived from the plasmid encoding the original antigen and showed that four distinct epitopes were recognized by the different antibodies. A 25-amino-acid peptide was used in competition ELISAs to confirm the location of the epitope recognized by two antibodies CE10 and AC6. Preliminary characterization of an NS-1/NS-2 fusion protein synthesized in insect cells using a baculovirus expression vector showed that this fusion protein is also localized within the nucleus; however, in contrast, the full-length NS-1 polypeptide is located within the cytoplasm.