Survivin is a tumor-associated antigen (TAA) that has significant potential for use as a cancer vaccine target. To identify survivin epitopes that might serve as targets for CTL-mediated, anti-tumor responses, we evaluated a series of survivin peptides with predicted binding to mouse H2-K(b) and human HLA-A*0201 antigens in peptide-loaded dendritic cell (DC) vaccines. H2-K(b)-positive, C57BL/6 mice were vaccinated using syngeneic, peptide-loaded DC2.4 cells. Splenocytes from vaccinated mice were screened by flow cytometry for binding of dimeric H2-K(b):Ig to peptide-specific CD8+ T cells. Two survivin peptides (SVN(57-64) and SVN(82-89)) generated specific CD8+ T cells. We chose to focus on the SVN(57-64) peptide because that region of the molecule is 100% homologous to human survivin. A larger peptide (SVN(53-67)), containing multiple class I epitopes, and a potential class II ligand, was able to elicit both CD8+ CTL and CD4+ T cell help. We tested the SVN(53-67) 15-mer peptide in a therapeutic model using a peptide-loaded DC vaccine in C57BL/6 mice with survivin-expressing GL261 cerebral gliomas. This vaccine produced significant CTL responses and helper T cell-associated cytokine production, resulting in a significant prolongation of survival. The SVN(53-67) vaccine was significantly more effective than the SVN(57-64) core epitope as a cancer vaccine, emphasizing the potential benefit of incorporating multiple class I epitopes and associated cytokine support within a single peptide.