One defining characteristic of Conus venom peptides is the high frequency of posttranslational modifications found. We report the discovery and initial characterization of three novel gamma-carboxyglutamic acid (Gla)-containing conotoxins, Gla-MrII, Gla-MrIII, and Gla-MrIV, isolated from the venom of the mollusc-hunting cone snail Conus marmoreus. Peptide Gla-MrII, a 50 amino acid residue peptide, carries eight cysteine residues arranged in a novel cysteine pattern, and five gamma-carboxyglutamic acid residues. The monoisotopic molecular mass was determined by electrospray ionization mass spectrometry to 5860.23 Da, consistent with the peptide having the cysteine residues disulphide-bonded and having a free acid C-terminus. Peptides Gla-MrIII and Gla-MrIV each contain two gamma-carboxyglutamic acid residues and share little sequence similarity to previously identified conotoxins. Both peptides contain four cysteine residues that are positioned in the linear sequence in a manner reminiscent of conotoxins belonging to cysteine scaffold superfamily T (scaffold T-1). Determination of the monoisotopic molecular masses revealed that Gla-MrIII is amidated at its C-terminus while Gla-MrIV has a free C-terminal acid. (C) 2004 Elsevier Inc. All rights reserved.