Three distinct antigenic determinants on the nucleoprotein (NP) of measles virus were localized. These epitopes were defined by three monoclonal antibodies, one of which recognized all measles virus field strains examined, whereas the other two were variable. A measles virus NP cDNA subclone representing 502 of the 525 amino acids was cloned into a bacterial expression vector plasmid (pRIT) and expressed as a Protein A-NP fusion protein in Escherichia coli. The expressed protein reacted with all three monoclonal antibodies. A series of NP gene deletion was constructed in order to locate the antigenic sites. The antigenic site identified on all measles virus strains studied, which was designated site I, was located between amino acids 122 and 150. The two variable epitopes were located at the C terminus of the protein (site II at 457 to 476; site III at 519 to 525). The structural and biological implications of these observations are discussed.