In a prior report (Int J Cancer 2006; 119: 339-348), we described a new vaccination strategy for squamous cell carcinoma (SCC). The vaccine was prepared by transfer of unfractionated DNA-fragments (25 kb) from KLN205 cells, a squamous carcinoma cell line (DBA/2 origin; H-2(d)) into LM cells, a highly immunogenic mouse fibroblast cell line (C3H/He origin; (H-2(k))). As only a small proportion of the transfected cell population was expected to have incorporated DNA segments that included genes specifying antigens associated with the squamous carcinoma cells, we devised a novel strategy to enrich the vaccine for immunotherapeutic cells. Enhanced immunity to squamous carcinoma was induced in tumor-bearing mice treated solely by immunization with the enriched vaccine, which translated into prolonged survival without toxicity. Here, we describe the characteristics of the cell populations infiltrating established squamous carcinomas undergoing regression in mice immunized with vaccines enriched for immunotherapeutic cells. The results indicated that CD8+ T cells were predominant and that T-regulatory cells (FoxP3+, CD4/CD25+, CD4/CD62L(high), CD4/CTLA-4e) were relatively deficient in the regressing tumors. Inflammatory infiltrates were not detected in various organs and tissues of mice immunized with the DNA-based vaccine.