Human bocavirus (HBoV) is a widespread respiratory virus. To improve diagnostic methods, we conducted immunoglobulin (Ig) G and IgM enzyme immunoassays with recombinant virus-like particles of HBoV as antigen. Acute-phase and follow-up serum samples from 258 wheezing children and single serum samples from 115 healthy adults in Finland were examined. Our assays had a sensitivity of 97% and a specificity of 99.5%. Of adults, 96% had immunity; none had an acute infection. Of 48 children with serologically diagnosed acute HBoV infections, 45 were viremic and 35 had virus in nasopharyngeal aspirates (NPAs). Of 39 HBoV NPA PCR-positive children co-infected with another virus, 64% had a serologically verified HBoV infection. HBoV caused illness of longer duration than rhinovirus and of equal severity to that of respiratory syncytial virus. Among children with bronchiolitis, >25% had acute HBoV infections. Accurate HBoV diagnosis requires serologic analysis or PCR of serum; PCR of NPAs alone is insufficient.