Recently, molecular screening for pathogenic agents has identified a partial genome of a novel parvovirus, called human bocavirus (HBoV). The presence of this newly described parvovirus correlated with upper and lower respiratory tract infections in children. Lower respiratory tract infections are a leading cause of hospital admission in children, and the etiological agent has not been identified in up to 39% of these cases. Using baculovirus expression vectors (BEVs) and an insect cell system, we produced virus-like particles (VLPs) of HBoV. The engineered BEVs express the HBoV capsid proteins stoichiometrically from a single open reading frame. Three capsid proteins assemble into the VLP rather than two proteins predicted from the HBoV genome sequence. The denatured capsid proteins VP1, VP2, and VP3 resolve on silver-stained sodium dodecyl sulfate-polyacrylamide gels as three bands with apparent molecular masses of 72 kDa, 68 kDa, and 62 kDa, respectively. VP2 apparently initiates at a GCT codon (alanine) 273 nucleotides downstream from the VP1 start site and 114 nucleotides upstream from the VP3 initiation site. We characterized the stable capsids using physical, biochemical, and serological techniques. We found that the density of the VLP is 1.32 g/cm(3) and is consistent with an icosahedral symmetry with approximately a 25-nm diameter. Rabbit antiserum against the capsid of HBoV, which did not cross-react with adeno-associated virus type 2, was used to develop enzyme-linked immunosorbent assays (ELISAs) for anti-HBoV antibodies in human serum. Using ELISA, we tested 404 human serum samples and established a range of antibody titers in a large U.S. adult population sample.