Stable transfection of tumor cells with IFN-α genes has been shown to result in abrogation of tumor establishment and induction of antitumor immunity. However, strategies suitable for the clinical application of IFN-α gene therapy for cancer have not been reported. In this study, we investigated two gene delivery systems capable of mediating the local paracrine production of high levels of biologically active IFN-α in murine tumor models: retroviral transduction of fibroblasts and particle-mediated transfection of tumor cells. In spite of the antiproliferative effects of IFN-α, it was possible to obtain stable retroviral producer cell lines and transduce a variety of murine tumor cells including syngeneic fibroblasts to stably secrete 2000–5000 U (40–100 ng) murine IFN-α/10 6 cells/24 h. IFN-α transduction of tumor cells abrogated tumorigenicity in establishment models and induced antitumor immunity in several murine tumor model systems. Importantly, IFN-α gene delivery using retrovirally transduced syngeneic fibroblasts was capable of suppressing the establishment of the poorly immunogenic TS/A mouse mammary adenocarcinoma and induced antitumor immunity. Particle-mediated transient transfection of tumor cells using the gene gun led to the production of up to 20000 U IFN-α/10 6 cells during the first 24 h and proved to be equally effective in suppressing establishment of TS/A adenocarcinoma and inducing antitumor immunity. These results suggest that retroviral transduction of autologous fibroblasts can serve as an effective gene delivery method for IFN-α gene therapy of cancer. Particle-mediated transfection of freshly isolated tumor cells may represent a clinically attractive alternative approach for nonviral gene delivery. Both strategies circumvent the difficulties in routinely establishing primary tumor cell lines from the vast majority of human cancers.