HIV-1 Gag protein represents a promising target of cellular immunity-based vaccines due to its immunogenicity and high conservation among diverse viral subtypes. Development of novel and effective Gag-targeted vaccine candidates inducing CD8(+) and CD4(+) T cell responses requires large scale pre-clinical testing in a small animal model. In this report, the MHC class I and II-restricted epitopes in the simian immunodeficiency virus (SIV) Gag protein recognized in C57Bl/6 and Balb/c mice were determined and characterized. In addition, using the newly defined epitopes, the relationship is described between the amount of plasmid DNA, volume of inoculate, and the extent of ensuing immune responses following intramuscular DNA immunization.