Many aspects of the presentation of viral peptides on MHC I for recognition by CD8+ T cells remain untested empirically having been extrapolated from simple model antigen systems or drawn from theory. To address these gaps, we have been using advanced mass spectrometry methods to analyze the presentation of many native vaccinia virus antigens by MHC I on mouse cells and correlating this information with the CD8+ T cell response during infection. Here we confirm and substantially extend our recent publication1 and show: 1) Presentation of viral peptides on MHC I during virus infection is complex and dynamic. 2) There are substantial cell type differences in the presentation of many epitopes, with some peptides differing in presentation level by more than 10-fold between DC-like and epithelial-like cell lines. 3) Most (~90%) of viral epitopes are generated from their source protein co-incident with expression. 4) The infected cell surface is poorly reflected in the anti-viral CD8+ T cell repertoire, with many highly abundant peptide-MHC complexes eliciting only weak or non-existent responses. 5) MHC-I binding algorithms fail to find many presented and immunogenic peptides. 6) For the first time we have determined the fraction of viral peptide-MHC complexes that are immunogenic, finding that nearly 70% of peptides presented from vaccinia virus elicit responses during infection. Together these data demonstrate that there are many surprises waiting in viral immunopeptidomes and there is an urgent need for more data if we are to manipulate this aspect of immunity to develop better vaccines.