Major hlstocompatlblllty complex(MHC) class I-restricted cytotoxlc T lymphocytes (CTL) recognize peptlde epltopes of protein antigens In a hierarchical fashion. We Investigated whether proteolytlc cleavage, in particular by proteasomes, Is Important in determlnlng epitope hierarchy. Using highly purified 20s proteasomes, we find preferred cleavage sites directly adjacent to the N-and C-terminal ends of the Immunodominant epltope of chicken ovalbumln, Ova257-264, while most of the subdominant epltope, Ova55-62, is destroyed by a major cleavage site located within this epltope. Moreover, we show that variations In amino acid sequences flanking these epltopes influence proteasomal cleavage patterns In parallel with the efficacy of their presentation. The results suggest that proteasomal cleavage within and adjacent to class I-restricted epltopes contrlbutes to their level of presentation.