Borna disease virus (BDV) is representative of the family of Bornaviridae in the order Mononegavirales (negative-stranded, non-segmented, enveloped RNA viruses). It is the causal agent for Borna disease, characterized as an encephalomyelitis (typical form) in a wide variety of domestic animals (from rodents to birds). Recent information shows the involvement of BDV in the pathogenesis of some human psychiatric disorders. The 8.9-kb viral antigenome codes for five major ORF. The third ORF codes for a 16-kDa protein (matrix protein) that is posttranslationally modified, yielding an N-linked glycoprotein. Our data show that the glycosylated matrix protein exists as a stable tetrameric structure detectable either by electrospray ionization or matrix-assisted laser-desorption ionization mass spectrometry. Under native conditions, the tetramer, with a relative molecular mass of 68 kDa, was isolated from a sediment-free brain suspension of a BDV-infected horse. The 68-kDa entity is stable in the presence of ionic and nonionic detergents but dissociates into subunits when heated. We found that the tetrameric matrix protein inhibits in vitro BDV infection in a dose-dependent manner. In contrast to inhibition of BDV infection with hydrophobic carbohydrate derivatives and protein-bound glycoconjugates, the glycosylated matrix protein is a very potent inhibitor of BDV infection, indicating that this protein represents an essential virus-specific membrane component for viral attachment.