Mature T lymphocytes comprise functionally distinct subsets with discrete roles in the regulation of the immune response. The cellular basis of the anti-tumor effect is now understood to involve the activation and expansion of tumor-specific cytotoxic T lymphocytes (CTL). To immuno-potentiate the generation of CTL, we have employed the biolistic system for the genetic immunization of mice. Here, we report the efficient generation of anti-H-2Kb allo-reactive CTL by particle acceleration-mediated genetic immunization of mouse spleen cells with H-2Kb DNA. The insertion and expression of exogenous gene into host spleen cells following in situ genetic inoculation to effect the generation of a cellular immune response may permit novel alternative strategies for immunotherapy.