The Norwalk Virus (NV) is the prototype strain of human caliciviruses that cause epidemic outbreaks of foodborne and waterborne gastroenteritis. These viruses do not grow in cell culture and the mechanisms of virus replication are obscure. The NV genome is a 7.7 kb ssRNA molecule that encodes three open reading frames (ORFs). The first ORF is a 1789 amino acid polyprotein that is processed into nonstructural proteins by a viral protease similar to the picornavirus 3C protease. Primary cleavage sites in the ORF1 polyprotein of two Norwalk-like viruses have been identified as QG dipeptides. We studied primary cleavage sites in the NV polyprotein and residues surrounding the scissile bond that are important in substrate recognition. A series of mutations were made at amino acids occupying positions implicated as important in cleavage site recognition for chymotrypsin-like viral proteases. We determined that effective processing at amino acid 398 to release the N-terminal p48 protein is necessary for proteolytic release of the p41 protein, that the P4 position N-terminal to the scissile bond is important for efficient processing, and that substitution of large hydrophobic residues were tolerated at this position. Finally, we defined the acidic residue of the 3CL(pro) catalytic site.