Dendritic cells (DC) pulsed with tumor-derived RNA are highly effective antigen-presenting cells (APC) that can induce cytotoxic T lymphocyte (CTL) responses and tumor immunity in vitro and in vivo. This study demonstrates that DC pulsed with RNA from tumor cells, including in vitro synthesized chicken ovalbumin (OVA) RNA, are more effective than DC pulsed with OVA peptides in stimulating OVA-specific CTL responses. DC pulsed with total or polyA⁺ RNA from OVA-expressing tumor cells were as effective as those pulsed with OVA peptides. The effectiveness of RNA was confirmed by showing that treatment with an OVA-specific antisense oligodeoxynucleotide and RNase H abrogated CTL induction, indicating that OVA RNA is responsible for sensitizing DC. Mice vaccinated with DC pulsed with RNA from OVA-expressing tumor cells were protected against tumor challenge, and in the B16/F10.9 melanoma model, DC pulsed with tumor-derived RNA significantly reduced lung metastases. The study also shows that RNA transcribed in vitro from cDNA cloned in a bacterial plasmid is highly effective in sensitizing DC, allowing the amplification of antigenic content from a small number of tumor cells. This expands the potential use of RNA-pulsed DC-based vaccines for patients with very small or microscopic tumors. The findings suggest that RNA-pulsed DC are a promising approach for inducing antitumor immunity, as they can effectively present antigens to T cells and elicit strong immune responses. The study highlights the potential of RNA-based vaccines in cancer immunotherapy.Dendritic cells (DC) pulsed with tumor-derived RNA are highly effective antigen-presenting cells (APC) that can induce cytotoxic T lymphocyte (CTL) responses and tumor immunity in vitro and in vivo. This study demonstrates that DC pulsed with RNA from tumor cells, including in vitro synthesized chicken ovalbumin (OVA) RNA, are more effective than DC pulsed with OVA peptides in stimulating OVA-specific CTL responses. DC pulsed with total or polyA⁺ RNA from OVA-expressing tumor cells were as effective as those pulsed with OVA peptides. The effectiveness of RNA was confirmed by showing that treatment with an OVA-specific antisense oligodeoxynucleotide and RNase H abrogated CTL induction, indicating that OVA RNA is responsible for sensitizing DC. Mice vaccinated with DC pulsed with RNA from OVA-expressing tumor cells were protected against tumor challenge, and in the B16/F10.9 melanoma model, DC pulsed with tumor-derived RNA significantly reduced lung metastases. The study also shows that RNA transcribed in vitro from cDNA cloned in a bacterial plasmid is highly effective in sensitizing DC, allowing the amplification of antigenic content from a small number of tumor cells. This expands the potential use of RNA-pulsed DC-based vaccines for patients with very small or microscopic tumors. The findings suggest that RNA-pulsed DC are a promising approach for inducing antitumor immunity, as they can effectively present antigens to T cells and elicit strong immune responses. The study highlights the potential of RNA-based vaccines in cancer immunotherapy.