We identified that the antigen preprocalcitonin (ppCT) is recognized on a human lung carcinoma by a cytotoxic T lymphocyte clone derived from autologous tumor-infiltrating lymphocytes. The antigenic peptide ppCT16–25 is encoded by the gene calcitonin-related polypeptide alpha (CALCA), which codes for CT and is overexpressed in several lung carcinomas compared with normal tissues. The ppCT peptide is derived from the C-terminal region of the signal peptide and is processed independently of proteasomes and the transporter associated with antigen processing (TAP)1/TAP2 heterodimeric complexes. Instead, processing occurs within the endoplasmic reticulum by a novel mechanism involving signal pepsidase (SP) and signal peptide peptidase (SPP). Although lung cancer cells bearing the ppCT16–25 epitope displayed low levels of TAP, restoration of TAP expression by interferon (IFN)-γ treatment or by TAP1/TAP2 gene transfer inhibited ppCT antigen presentation. Thus, the ppCT16–25 human tumor epitope requires low TAP expression for efficient presentation. These results indicate that emerging SP-generated peptides represent alternative T cell targets that permit cytotoxic T lymphocytes to destroy TAP-impaired tumors, a process that helps to overcome tumor escape from CD8+ T cell immunity. Additionally, our data suggest that ppCT is a promising candidate for cancer immunotherapy.