Human and mouse genomes contain more than 20 related genes encoding diverse type I interferons (IFNs- alpha/beta), cytokines that are crucial for resistance of organisms against viral infections. Although the amino acid sequences of various IFN-alpha/beta subtypes differ markedly, they are all considered to share a common three-dimensional structure and to bind the same heterodimeric receptor, composed of the IFNAR-1 and IFNAR-2 subunits. Analysis of available mammalian IFN-beta sequences showed that they all carry 1 to 5 predicted N-glycosylation sites. Murine IFN-beta contains three predicted N-glycosylation sites (Asn29, Asn69, Asn76), one of which (Asn29) is located in the AB loop, in a region predicted to interact with the type I IFN receptor. The aim of this work was to test if this site is indeed N-glycosylated and if this glycosylation would affect IFN antiviral activity. We showed that all three N-glycosylation sites predicted from the sequence, including Asn29, carry N-linked sugars. Mutation of individual N-glycosylation sites had a weak negative influence on IFN antiviral activity. In contrast, the complete loss of glycosylation dramatically decreased activity. Our data suggest that interaction of murine IFN-beta with the IFNAR could locally differ from that of human IFN-alpha2 and human IFN-beta.