The successful induction of T cell-mediated protective immunity against poorly immunogenic malignancies remains a major challenge for cancer immunotherapy. Here, we demonstrate that the induction of tumor-protective immunity by IL-12 in a murine neuroblastoma model depends entirely on the CXC chemokine IFN-gamma-inducible protein 10 (IP-10). This was established by in vivo depletion of IP-10 with mAbs in mice vaccinated against NXS2 neuroblastoma by gene therapy with a linearized, single-chain (sc) version of the heterodimeric cytokine IL-12 (scIL-12). The efficacy of IP-10 depletion was indicated by the effective abrogation of scIL-12-mediated antiangiogenesis and T cell chemotaxis in mice receiving s.c. injections of scIL-12-producing NXS2 cells. These findings were extended by data demonstrating that IP-10 is directly involved in the generation of a tumor-protective CD8+ T cell-mediated immune response during the early immunization phase. Four lines of evidence support this contention: First, A/J mice vaccinated with NXS2 scIL-12 and depleted of IP-10 by two different anti-IP-10 mAbs revealed an abrogation of systemic-protective immunity against disseminated metastases. Second, CD8+ T cell-mediated MHC class I Ag-restricted tumor cell lysis was inhibited in such mice. Third, intracellular IFN-gamma expressed by proliferating CD8+ T cells was substantially inhibited in IP-10-depleted, scIL-12 NXS2-vaccinated mice. Fourth, systemic tumor protective immunity was completely abrogated in mice depleted of IP-10 in the early immunization phase, but not if IP-10 was depleted only in the effector phase. These findings suggest that IP-10 plays a crucial role during the early immunization phase in the induction of immunity against neuroblastoma by scIL-12 gene therapy.