Dendritic cells (DC) are professional antigen-presenting cells that play a pivotal role in the induction of immunity. Ex vivo–generated, tumor antigen–loaded mature DC are currently exploited as cancer vaccines in clinical studies. However, antigen loading and maturation of DC directly in vivo would greatly facilitate the application of DC-based vaccines. We have previously shown that in situ tumor destruction by ablative treatments efficiently delivers antigens for the in vivo induction of antitumor immunity. In this article, we show that although 20% of the draining lymph node DCs acquire intratumorally injected model antigens after in situ cryoablation, only partial protection against a subsequent tumor rechallenge is observed. However, we also show that a combination treatment of cryoablation plus TLR9 stimulation via CpG-oligodeoxynucleotides is far more effective in the eradication of local and systemic tumors than either treatment modality alone. Analysis of the underlying mechanism revealed that in situ tumor ablation synergizes with TLR9 stimulation to induce DC maturation and efficient cross-presentation in tumor-bearing mice, leading to superior DC function in vivo. Therefore, in situ tumor destruction in combination with CpG-oligodeoxynucleotide administration creates a unique “in situ DC vaccine” that is readily applicable in the clinic. (Cancer Res 2006; 66(14): 7285-92)