We have previously described a method for adoptive immunotherapy of cancer based on antigen-specific T(h)1 cells. However, efficient induction of anti-tumor responses using T(h)1 cells remains a formidable challenge, especially for MHC class II-negative tumors. In the present study, we sought to develop a novel strategy to eradicate established tumors of the MHC class II-negative, ovalbumin (OVA)-expressing EG-7 cells. Tumor-bearing mice were intradermally treated with OVA-specific T(h)1 cells, combined with the model tumor antigen (OVA), near the tumor-draining lymph node (DLN). We found that tumor growth was significantly inhibited by this strategy and approximately 50-60% of tumor-bearing mice were completely cured. Tumor eradication was crucially dependent on the generation of OVA/H-2K(b)-specific CTLs in the tumor DLNs and tumor site. The injected T(h)1 cells were mainly distributed in tumor DLNs, where they vigorously proliferated and enhanced the activation of dendritic cells. Strikingly, we also found that the accumulation of CD4(+)CD25(+) regulatory T cells (Tregs) was significantly inhibited in tumor DLNs by T(h)1 cell adjuvant therapy and this abrogation was associated with IFNgamma secreted by T(h)1 cells. These results identify T(h)1 cell adjuvant therapy combined with tumor vaccination as a novel approach to the treatment of human cancer.