The epitopes recognized by CD8+ cytotoxic T lymphocytes (CTL) are generated from cytosolic proteins by proteolytic processing. CD8+ a3 cytotoxic T lymphocytes (CTL) recognize short peptides (epitopes) associated with specific alleles of the class I major histocompatibility complex (1). The peptide epitopes are mainly generated from cytosolic proteins by proteolysis, a process believed to involve the multicatalytic proteosome complex (2-8). The influence of sequences flanking CTL epitopes on the proteolytic processing and presentation of these epitopes remains controversial (9-14). Moving CTL epitopes into new positions within a protein in some cases prevents (9) and in other cases has no effect on (10, 11) presentation of the epitope. A profound effect on presentation was observed when an epitope was expressed with different truncations of the natural flanking sequences (12). However, only one of a series of substitutions made to the amino acid residues flanking a CTL epitope was shown to effect presentation (13,14). The subdominance of a CTL epitope has been attributed to the inability of proteosomes to generate the peptide from the parent protein (15), although recently the subdominance of this epitope has been attributed to low affinity of the peptide for its restriction element (16). Although some of these data may illustrate that certain flanking sequences prevent proteolytic generation of an epitope, it is not clear whether the presence of any specific natural flanking sequences is needed to direct the activity of proteolytic processing enzymes.To determine whether any natural sequences outside CTL epitopes are a requirement for class I processing, a recombinant vaccinia virus (VV) was constructed that coded for a single artificial polyepitope protein containing nine class I-restricted CD8+ CTL epitopes in sequence (see Fig.