Cytotoxic T lymphocytes (CTLs) are the most powerful weapon of the immune system to eliminate cells infected by intracellular parasites or tumors. However, very often, escape mechanisms overcome CTL immune surveillance by impairing the classical HLA class I antigen-processing pathway. Here, we describe a strategy for CTL activation based on the ability of Tat to mediate transcellular delivery of viral proteins encompassing HLA class I-restricted epitopes. In this system, the recombinant protein TAT-NpFlu containing the transduction domain of Tat of human immunodeficiency virus type 1 fused to the amino acid region 301 to 498 of the nucleoprotein of influenza A virus is proven to sensitize different human cells to lysis by HLA-B27-restricted, Flu 383-391-specific CTL lines. The fusion protein is processed very effectively, since a comparable biological effect is obtained with an amount of protein between 1 and 2 orders of magnitude lower than that of the synthetic peptide. Interestingly, while part of TAT-NpFlu undergoes fast and productive cleavage, a large amount of it remains intact for up to 24 h. Confocal microscopy shows that TAT-NpFlu accumulates in the trans-Golgi network (TGN), where it starts to be detectable 1 h after transduction. Using TAT-NpFlu mutants and hybrid constructs, we demonstrate that enrichment in the TGN occurs only when the carboxy-terminal region of NpFlu (amino acids 400 to 498) is present. These data disclose an unconventional route for presentation of epitopes restricted for HLA class I molecules.