Direct determination of the amino acid sequence of VP60 from rabbit hemorrhagic disease virus is impeded by the presence of a blocked N-terminus. Chemical cleavage of VP60 using cyanogen bromide allowed the identification and purification of two oligopeptides showing identical amino acid composition, one of which had its amino terminus blocked. Automated sequential degradation of the unblocked CNBr- peptide yielded the amino acid sequence EGKARTAPQGEAA. This sequence is identical to the deduced amino acid sequence following the first AUG codon found at position +10 at the 5'-end of the 2.4 kb subgenomic mRNA. These data favor the hypothesis that this viral polypeptide is mainly produced from the subgenomic mRNA and not from the genomic RNA by processing of the putative polyprotein generated from the major open reading frame.