An excitatory peptide, di16a, with 49 amino acids and 10 cysteine residues was purified and characterized from the venom of Conus distans. Five AA residues were modified: one gamma-carboxyglutamate (Gla), and four hydroxyproline (Hyp) residues. A cDNA clone encoding the precursor for the peptide was characterized; the peptide has a novel cysteine framework and a distinctive signal sequence that differs from any other conotoxin superfamily. The peptide was chemically synthesized and folded, and synthetic and native materials were shown to co-elute. Injection of the synthetic peptide causes a hyperexcitable phenotype in mice greater than 3 weeks of age at lower doses, and lethargy at higher doses. The peptide defines both a previously uncharacterized gene superfamily of conopeptides, and a new Cys pattern with three vicinal Cys residues.