The second matrix (M2) gene of avian metapneumovirus subgroup C (aMPV-C) contains two overlapping open reading frames (ORFs), encoding two putative proteins, M2-1 and M2-2. Both proteins are believed to be involved in viral RNA transcription or replication. To further characterize the function of the M2-2 protein in virus replication, the non-overlapping region of the M2-2 ORF was deleted from an infectious cDNA clone of the aMPV-C strain, and a viable virus was rescued by using reverse genetics technology. The recombinant virus, raMPV-C ΔM2-2, was characterized in vitro and in vivo. In Vero cells, raMPV-C ΔM2-2 replicated slightly less efficiently than the parental virus, 10-fold reduction at 48-h post-infection. The raMPV-C ΔM2-2 virus induced typical cytopathic effects (CPE) that were indistinguishable from those seen with the parental virus infection. In specific-pathogen-free (SPF) turkeys, raMPV-C ΔM2-2 was attenuated and caused no clinical signs of disease. Less than 20% of the inoculated birds shed detectable virus in tracheal tissue during the first 5 days post-infection, and no virus shedding was detected afterward. Forty percent of infected birds produced a weak antibody response at 14 days post-infection. Upon challenge with a virulent aMPV-C strain, more than 80% of the raMPV-C ΔM2-2-inoculated birds showed typical disease signs and virus shedding in tracheal tissue. These results suggest that the M2-2 protein of aMPV-C virus is not essential for virus replication in vitro, but is required for sufficient virus replication to maintain pathogenicity and immunogenicity in the natural host.