In our efforts for cloning novel I(2)-superfamily conotoxins using the signal peptide sequence, we identified a novel conotoxin Lt12.4 from Conus litteratus. This gene has a framework XII (-C-C-C-C-CC-C-C-), which is distinct from the cysteine pattern I(2)-superfamily conotoxin (-C-C-CC-CC-C-C-). Subsequently, we found the signal peptide sequence of Lt12.4 by 5'-RACE. Using this new sequence, we identified another five novel conotoxins with this cysteine pattern from four Conus species (Conus eburneus, Conus imperialis, Conus marmoreus, and C. litteratus). These novel conotoxins have the same cysteine pattern as the reported Gla-TxX and Gla-MII, and may contain Gla residues. Furthermore, they have the highly conserved signal peptide and hypervariable mature peptide sequences, and widely exist in Conus species. Therefore, it could be defined as a new superfamily of E-conotoxins.