Human metapneumovirus (hMPV) strains are classified into two genetic groups, A and B, each of which is further divided in two genetic subgroups, A1, A2, B1 and B2. hMPV encodes two major surface glycoproteins, the fusion (F) and attachment (G) proteins, which may be immunogenic and protective antigens. Although the amino acid sequences of hMPV F protein are highly conserved, those of the G protein are highly variable with low amino acid identity between the two groups. To address the antigenic variation between the genetic subgroups, we developed an immunofluorescence assay (IFA) method using Trichoplusia ni (Tn5) insect cells infected with each recombinant baculovirus-expressed hMPV G (Bac-G) protein of the four genetic subgroups. The titre of each antibody to the four Bac-G proteins was measured by the IFA in 12 paired serum samples obtained from children infected with hMPV of each genetic subgroup. Although 11 of the 12 acute-phase serum samples in paired samples were negative for the antibody to any Bac-G proteins, all of the convalescent-phase serum samples in those paired samples were positive for the antibody to only one of the four Bac-G proteins of the infecting genotype of hMPV. Since the antibody response to hMPV G protein was transient and genetic subgroup-specific without cross-reactivity, four genetic subgroups on the basis of hMPV G protein could be identified as different serotypes. This assay may be useful for the study of immune responses of humans to different hMPV strains, especially for clarifying the risk of reinfection with hMPV.