Peptide splicing is a novel mechanism of production of peptides relying on the proteasome and involving the linkage of fragments originally distant in the parental protein. Peptides produced by splicing can be presented on class I molecules of the major histocompatibility complex and recognized by cytolytic T lymphocytes. Here, we describe a new antigenic peptide, which is presented by HLA-A3 and comprises two non-contiguous fragments of the melanoma differentiation antigen gp100PMEL17 spliced together in the reverse order to that in which they appear in the parental protein. Contrary to the previously described spliced peptides, which are produced by the association of fragments of 3 to 6 amino acids, the peptide described here results from the ultimate association of an 8-amino acid fragment with a single arginine residue. As described before, peptide splicing takes place in the proteasome by transpeptidation involving an acyl-enzyme intermediate linking one of the peptide fragment to a catalytic subunit of the proteasome. Interestingly, we observe that the peptide causing the nucleophilic attack on the acyl-enzyme intermediate must be at least three-amino acid long in order to give rise to a spliced peptide. The spliced peptide produced from this reaction therefore bears an extended C-terminus that needs to be further trimmed in order to produce the final antigenic peptide. We show that the proteasome is able to perform the final trimming step required to produce the antigenic peptide described here.