Human parainfluenza virus type 3 (hPIV3) is a respiratory tract pathogen. The current study aimed to investigate immunodominant regions of hPIV3 nucleocapsid (N) protein by using monoclonal antibodies (mAbs) raised against recombinant N protein and human serum specimens from hPIV3-infected individuals. A panel of murine mAbs was generated following immunization with yeast-expressed hPIV3 N protein self-assembled to nucleocapsid-like particles. All mAbs recognized native viral nucleocapsids in hPIV3-infected cells as confirmed by an indirect immunofluorescence analysis. Antigenic sites recognized by the mAbs were mapped using recombinant overlapping N protein fragments. One major immunodominant site was identified in the carboxy-terminal region (amino acids aa 397-486) of hPIV3 N protein. Further analysis with smaller N protein fragments and a synthetic peptide revealed one linear epitope representing aa 437-446 of the N protein located within this antigenic site. This epitope was reactive with 46% of hPIV3 IgG-positive sera. These results suggest that the above antigenic site on the N protein is important in eliciting a humoral immune response against hPIV3.