The 454-amino acid nucleocapsid (N) protein of mouse hepatitis virus (MHV) binds the leader RNA sequence located at the 5′ ends of all plus-sense genomic and subgenomic viral mRNAs. Purified N protein was cleaved with formic acid to determine which domain interacts with the leader RNA sequence. Incubation at 42 °C resulted in partial cleavage into two fragments of M rs of approximately 32K and 37K and three fragments of 17K, 16K and 14K. Incubation at 56 °C resulted in complete cleavage yielding only the three lower molecular mass products. Both the 32K and 37K partial cleavage products and one of the complete cleavage products bind MHV leader RNA, suggesting that the central region of the N protein contains the RNA-binding domain. Monoclonal antibody mapping of the cleavage products confirmed that the MHV leader RNA binding domain is contained within the central 140-amino acid fragment, comprising amino acids 169 to 308. Analysis of the amino acids within this domain indicates no similarity to any previously described RNA-binding protein, suggesting that N protein may possess a unique RNA-binding motif.