The majority of MHC class I epitopes is generated through the ubiquitin-proteasome system. In the present study, we have analyzed the proteasome-dependent generation of the IE pp89 MCMV-derived H-2L(d) epitope by both in vitro and in vivo experiments. As revealed by cytotoxic T-cell assays, the pp89 9mer epitope was generated with high fidelity from the recombinant IE pp89 by 20S proteasomes. In vitro processing showed that the recombinant pp89 was rapidly degraded by 20S proteasomes. Analysis of cell lysates under conditions that allowed detection of polyubiquitinated proteins provided no evidence for the presence of ubiquitin-pp89-conjugates in vivo. These findings suggest a ubiquitin-independent mechanism of proteasomal degradation for pp89. (c) 2007 Elsevier Inc. All rights reserved.