Recent studies have defined vaccinia virus (VACV)-specific CD8+ T cell epitopes in mice and humans. However, little is known about the epitope specificities of CD4+ T cell responses. In this study, we identified 14 I-Ab-restricted VACV-specific CD4+ T cell epitopes by screening a large set of 2146 different 15-mer peptides in C57BL/6 mice. These epitopes account for ∼20% of the total anti-VACV CD4+ T cell response and are derived from 13 different viral proteins. Surprisingly, none of the CD4+ T cell epitopes identified was derived from VACV virulence factors. Although early Ags were recognized, late Ags predominated as CD4 + T cell targets. These results are in contrast to what was previously found in CD8+ T cells responses, where early Ags, including virulence factors, were prominently recognized. Taken together, these results highlight fundamental differences in immunodominance of CD4+ and CD8+ T cell responses to a complex pathogen. Copyright © 2007 by The American Association of Immunologists, Inc.