The melanosomal protein tyrosinase is considered as a target of specific immunotherapy against melanoma. Two tyrosinase‐derived peptides are presented in association with HLA‐A2.1 [Wölfel et al., Eur. J. Immunol., 24, 759–764 (1994)]. Peptide 1‐9 (MLLAVLYCL) is generated from the putative signal sequence. The internal peptide 369‐377 is posttranslationally converted at residue 371, and its presentation is dependent on functional TAP transporters and proteasomes [Mosse et al., J. exp. Med.187, 37–48 (1998)]. Herein, we report on the processing and transport requirements for the signal sequence‐derived peptide 1‐9 that were studied in parallel to those for peptide 369‐377. After infection of TAP‐deficient (T2) and TAP‐positive (T1) cells with a Modified Vaccinia Ankara construct carrying the human tyrosinase gene (MVA‐hTyr), we found that recognition by CTL against peptide 1‐9 did not require TAP function as opposed to recognition by CTL against peptide 369‐377. When target cells with intact processing and transport functions were infected with MVA‐hTyr, lysis by CTL against peptide 1‐9 was not impaired by lactacystin, a specific inhibitor for the proteasome, whereas lysis by CTL against peptide 369‐377 was completely abrogated. Taken together, peptide 1‐9 derived from the signal sequence of tyrosinase is presented in a TAP‐independent fashion and does not require proteasomes for processing. Cellular immune responses against this hydrophobic peptide can be monitored with lymphokine spot assays as documented in the case of a patient with metastatic melanoma, in whom we observed a preferential T‐cell response against tyrosinase peptide 1‐9 subsequent to chemoimmunotherapy. Independence of cytosolic processing and transport pathways and potentially enhanced expression levels make signal sequence‐derived peptides and their carrier proteins important candidates for specific immunotherapy.Int. J. Cancer 88:432–438, 2000. © 2000 Wiley‐Liss, Inc.