Respiratory tract infections are a leading cause of morbidity and mortality worldwide. Human metapneumovirus (HMPV) is a recently discovered respiratory pathogen of the Paramyxovirus family in the Metapneumovirus genus. HMPV was first isolated from young children in The Netherlands with respiratory illness similar to human respiratory syncytial virus (RSV) infection. Epidemiological data indicates that HMPV co-circulates with RSV in the community. Few immunological tools are available to study the virological features of HMPV infection, thus current studies rely on reverse-transcription (RT) polymerase chain reaction (PCR) for detection. In this study, we examine serological cross-reactivity of RSV, HMPV and other Metapneumovirus members, i.e. avian metapneumovirus (AMPV), and show that polyclonal and monoclonal antibodies reactive to a conserved region in AMPV nucleoprotein (N) cross-react with HMPV N protein, but not with RSV N protein by ELISA, Western blot and immunohistochemical assays. In addition, we show that HMPV infection in the lungs of BALB/c mice can be detected using anti-N protein antibody. These reagents provide new tools and methods for investigating HMPV infection, for differentiating HMPV from RSV infection, and may be useful for characterizing potential links between HMPV with other respiratory complications.