Abstract: Based on their role for the induction of T-cell responses, dendritic cells (DCs) are popular candidates in cancer vaccine development. We established a novel single-step intracellular delivery of peptide/poly(I:C) complexes for antigen loading and Toll-like receptor-3 (TLR3)-mediated maturation of human DCs using a cell-penetrating peptide (tat49–57: RKKRRQRRR) as delivery vector. Towards this end, a cationic tat-sequence was fused with an antigenic, major histocompatibility complex (MHC) class I-binding melanoma epitope (Melan-A/Mart-1 sequence: ELAGIGILTV) and then mixed with negatively charged poly(I:C) dsRNA to form peptide/nucleic acid complexes. Flow cytometry and confocal laser scanning microscopy confirmed intracellular localization of TLR3 in monocyte-derived immature DCs (iDCs). Peptide/poly(I:C) complexes were readily internalized by iDCs without negatively affecting cell viability. They induced DC maturation and secretion of bioactive interleukin (IL)-12p70. When peptide/poly(I:C) complex-loaded DCs were used for autologous T cell stimulation, epitope-specific interferon-gamma secretion was quantitatively superior in comparison to peptide-loaded DCs matured by a cytokine cocktail, as detected by enzyme-linked immunospot assays. Thus, complexes of cationic antigenic peptides and poly(I:C) might be of great utility for a TLR3-mediated DC maturation and intracellular peptide targeting in a single step. Resulting DCs induce a strong expansion/activation of antigen-specific T cells in the context of an IL-12p70 secretion.