A major peptide, de13a from the crude venom of Conus delessertii collected in the Yucatan Channel, Mexico, was purified. The peptide had a high content of posttranslationally modified amino acids, including 6-bromotryptophan and a nonstandard amino acid that proved to be 5-hydroxylysine. This is the first report of 5-hydroxylysine residues in conotoxins. The sequence analysis, together with cDNA cloning and a mass determination (monoisotopic mass of 3486.76 Da), established that the mature toxin has the sequence DCOTSCOTTCANGWECCKGYOCVNKACSGCTH, where O is 4-hydroxyproline, W 6-bromotryptophan, and K 5-hydroxylysine, the asterisk represents the amidated C-terminus, and the calculated monoisotopic mass is 3487.09 Da. The eight Cys residues are arranged in a pattern (C-C-C-CC-C-C-C) not described previously in conotoxins. This arrangement, for which we propose the designation of framework #13 or XIII, differs from the ones (C-C-CC-CC-C-C and C-C-C-C-CC-C-C) present in other conotoxins which also contain eight Cys residues. This peptide thus defines a novel class of conotoxins, with a new posttranslational modification not previously found in other Conus peptide families.