Chimeric peptides in which the optimal H-2d mouse hepatitis virus nucleocapsid (pN) and human immunodeficiency virus type 1 (p18) epitopes, separated by 38, 7, or 2 amino acids, were expressed from a single open reading frame by using recombinant vaccinia viruses to analyze antigen processing of proximal class I-restricted epitopes. Recognition of the carboxy-terminal Dd-restricted p18 epitope was independent of the amino-terminal flanking residues. By contrast, proximity of the carboxy-terminal epitope decreased recognition of the amino-terminal Ld-restricted pN epitope. Immunization resulted in the induction of both p18- and pN-specific antiviral cytotoxic T lymphocytes, irrespective of the number of amino acids separating the epitopes.