The use of recombinant and synthetic vaccines in the treatment of cancer has recently been explored using model tumor associated antigens (TAA), many of which do not model the immunological state of affairs in which the TAA is expressed by normal tissues. One potentially useful model Ag is β-galactosidase (β-gal). Because the activity of this enzyme is so easily detectable, this gene has been inserted into a large number of recombinant viruses and tumors useful to the cancer vaccinologist. In addition, numerous transgenic mouse colonies that have tissue-specific expression of β-gal have been developed, enabling the modeling of tolerance to “self” Ags. Since most of these mice have an H-2^bbackground, we generated cytotoxic T lymphocytes (CTL) capable of recognizing β-gal-expressing tumor cells of C57BL\6 origin and have determined that their restriction element is the K^bmolecule. Using an allele-specific epitope forecast to generate a panel of candidate peptides, we have determined that the K^b-restricted sequence is DAPIYTNV and corresponds to amino acids 96–103 of the intact β-gal molecule. A recombinant vaccinia virus (rVV-ES β-gal_96–103) was constructed that encoded the peptide epitope preceded by an endoplasmic reticulum insertion signal sequence. Tumor cells infected with this rVV were recognized by the original CTL that had been used to identify the epitope. Furthermore, splenocytes of mice immunized with a rVV encoding the full-length β-gal molecule and restimulated with the DAPIYTNV peptide specifically recognized tumor cells expressing β-gal. The identification of this immunogenic β-gal sequence enables the modeling of immunization strategies in animal models of malignant disease in which the target antigen is a “self” protein.