During translation of Murine hepatitis virus (MHV-A59) ORF1a, p28, the N-terminal polypeptide is cleaved from the growing polypeptide chain. Amino terminal radiosequencing of the resulting downstream cleavage product demonstrated that cleavage occurs between Gly247 and Val248. Site directed mutagenesis of amino acids surrounding the p28 cleavage site revealed that substitutions of Arg246 (P2) and Gly247 (P1) nearly eliminated cleavage of p28. Single amino acid substitutions of other residues between P7 and P2' were generally permissive for cleavage although a few changes did greatly reduce proteolysis. The amino acids around the p28 cleavage site represent a new sequence recognized by a virus encoded papain-like proteinase.