Human metapneumovirus (hMPV) is a newly identified respiratory virus associated with respiratory tract infection in both adults and children. Previous reports showed that infection of hMPV appeared to be ubiquitous. To determine the seroprevalence of hMPV, a total of 576 human sera from patients in Saskatchewan, Canada, were screened by enzyme-linked immunosorbent assay (ELISA) based on expression of the nucleocapsid (N) protein of hMPV in recombinant baculovirus. The recombinant N protein with a molecular mass of 43.5 kDa was abundantly produced in insect cells. Moreover, the recombinant N proteins of the prototype viruses for the two major groups of hMPV have cross-antigenicity. The seropositive rate for each age group was 13.5% (13/96) (0-5 years), 26.1% (25/96) (6-10 years), 32.3% (31/96) (11-15 years), 99.0% (95/96) (16-30 years), 91.7% (88/96) (31-60 years), and 93.8% (90/96) (61+ years), respectively. The data indicated that exposure to hMPV is a common phenomenon. The ELISA based on recombinant baculovirus produced N protein of hMPV provides a useful tool for seroepidemiological study of this virus.