This investigation was based on the hypothesis that weakly immunogenic, breast cancer-associated Ags, the products of mutant or dysregulated genes in the malignant cells, will be expressed in a highly immunogenic form by semiallogeneic IL-2-secreting fibroblasts transfected with DNA from breast cancer cells. (Classic studies indicate that transfection of genomic DNA can stably alter both the genotype and the phenotype of the cells that take up the exogenous DNA.) To investigate this question, we transfected LM mouse fibroblasts (H-2k) modified to secrete IL-2 with genomic DNA from a breast adenocarcinoma that arose spontaneously in a C3H/He mouse (H-2k). To increase their nonspecific immunogenic properties, the fibroblasts were also modified before transfection to express allogeneic MHC determinants (H-2Kb). Afterward, the IL-2-secreting semiallogeneic cells were cotransfected with DNA from the spontaneous breast neoplasm, along with a plasmid (pHyg) conferring resistance to hygromycin. Pooled colonies of hygromycin-resistant cells were then tested in C3H/He mice for their immunotherapeutic properties against the growth of the breast neoplasm. The results indicated that tumor-bearing mice immunized with the transfected cells survived significantly longer than mice in various control groups. Similar beneficial effects were seen in C57BL/6 mice injected with a syngeneic breast carcinoma cell line (EO771) and semiallogeneic, IL-2-secreting fibroblasts transfected with DNA from EO771 cells. The immunity was mediated by CD8+ T cells since immunized mice depleted of CD8+ cells failed to resist tumor growth.