We examined whether radiotherapy (RT) could enhance the efficacy of dendritic cell (DC)-based immunotherapy of cancer. Mice bearing s.c. D5 melanoma or MCA 205 sarcoma tumors were treated with intratumoral (i.t.) injections of bone marrow-derived unpulsed DCs in combination with local fractionated tumor irradiation. DC administration alone slightly inhibited D5 tumor growth and had no effect on MCA 205. RT alone caused a modest inhibition of both tumors. DC administration combined with RT inhibited D5 and MCA 205 tumor growth in an additive and synergistic manner, respectively. In both tumor models, RT intensified the antitumor efficacy of DC administration independent of apoptosis or necrosis within the tumor mass. Combination treatment of i.t. DCs plus RT was superior to s.c. injections of tumor lysate-pulsed DCs plus interleukin 2 in inhibiting D5 tumor growth and prolonging survival of mice. Splenocytes from mice treated with i.t. DCs plus RT contained significantly more tumor-specific, IFN-gamma-secreting T cells compared with control groups. Moreover, adoptive transfer of these splenocytes mediated significant tumor regression in mice bearing established pulmonary metastases. Combined treatment followed by resection of residual s.c. tumor conferred protective immunity against a subsequent i.v. tumor challenge. Furthermore, i.t. DC plus RT treatment of s.c. tumor in mice bearing concomitant pulmonary metastases resulted in a significant reduction of lung tumors. i.t. DC administration combined with RT induces a potent local and systemic antitumor response in tumor-bearing mice. This novel regimen may be beneficial in the treatment of human cancers.