Background We previously have shown that nona-arginine protein transduction domain (R9-PTD) induced efficient protein-antigen (Ag) transduction of dendritic cells (DCs) in vitro, resulting in the efficient induction of strong Ag-specific immune responses mediated by CD8+ and CD4+ T cells and in superior antitumour effects in vivo in cancer-bearing mice. Objectives The Ag-specific immune responses caused by intradermal (i.d.) injections of R9-PTD-containing protein Ags without DC preparation were investigated. We also investigated the antitumour effects by intratumoral (i.t.) injections of rR9-containing protein Ags. Methods Synthesized SIINFEKL peptide, or recombinant ovalbumin fusion proteins (rOVA, rR9-OVA), were directly injected into abdominal skin in naïve C57BL/6 mice. OVA-specific cytotoxic T lymphocyte (CTL) activity, serum IgG titre and cytokine profiles were investigated. Histopathological analyses were also performed. In a cancer vaccination model, EG.7 (OVA-cDNA transfectants thymoma) cells were inoculated intradermally in C57BL/6 mice, and the antitumour effects were evaluated by i.t. injections of rR9-OVA in a treatment setting. Results i.d. injections of rR9-OVA into naïve C57BL/6 mice elicited OVA-specific CTLs and produced IgG2-dominant immunoglobulin. The i.d. injections of rR9-OVA also induced inflammatory cell infiltrates containing neutrophils, monocytes and lymphocytes, as well as production of inflammatory cytokines such as interferon (IFN)-gamma, interleukin-2 and IFN-inducible protein 10, with presenting SIINFEKL epitopes on major histocompatibility complex (MHC) class I molecules at the injection area. i.t. injections of rR9-OVA into EG.7 tumour mass significantly suppressed tumour growth, and these effects were completely abrogated by the depletion of CD8+ T cells. These antitumour effects were superior to those elicited by i.t. injections of rR9-OVA-treated DCs. Conclusions i.d. injections of rR9-containing immunogenic Ag without adjuvants simultaneously induce dual immunological effects: the induction of Tc1- and Th1-dominant immune responses, and the induction of inflammatory and CTL-mediated immune responses at the injection area by expressing Ag epitopes on MHC class I molecules as targets. This simple vaccination approach with R9-PTD-containing fusion proteins might be useful as prophylactic immunotherapy for cancer or infectious diseases.