Most of the peptides presented by major histocompatibility complex (MHC) class I molecules require processing by proteasomes. Tripeptidyl peptidase II (TPPII), an aminopeptidase with endoproteolytic activity, may also have a role in antigen processing. Here, we analyzed the processing and presentation of the immunodominant human immunodeficiency virus epitope HIV-Nef(73–82) in human dendritic cells. We found that inhibition of proteasome activity did not impair Nef(73–82) epitope presentation. In contrast, specific inhibition of TPPII led to a reduction of Nef(73–82) epitope presentation. We propose that TPPII can act in combination with or independent of the proteasome system and can generate epitopes that evade generation by the proteasome-system.