The coronavirus nucleocapsid protein (N) is involved in encapsidation and packaging of viral RNA. In this study we investigated the ability of the bovine coronavirus (BCV) N protein to interact with RNA. Histidine-tagged BCV N (his-N) protein was expressed in bacteria. A filter binding assay was established to quantitatively measure the binding efficiency of purified his-N to different RNAs. The results indicate that bacterially expressed N bound both BCV and mouse hepatitis coronavirus (MHV) RNAs. Binding to in vitro generated BCV and MHV RNA transcripts was significantly higher than binding to a non-coronavirus RNA. Similar binding efficiencies were measured for a BCV defective genome, pDrep, and a transcript that contained the MHV packaging signal. Interestingly, the entire MHV DI, pMIDI-C, was bound at a higher efficiency than the packaging signal alone. This is one of the first reports to show that N interacts with the MHV packaging signal.