AbstractThe I‐conotoxin superfamily (I‐Ctx) is known to have four disulfide bonds with the cysteine arrangement C‐C‐CC‐CC‐C‐C, and the members inhibit or modify ion channels of nerve cells. Recently, Olivera and co‐workers (FEBS J. 2005; 272: 4178–4188) have suggested that the previously described I‐Ctx should now be divided into two different gene superfamilies, namely, I1 and I2, in view of their having two different types of signal peptides and exhibiting distinct functions. We have revisited the 28 entries presently grouped as I‐Ctx in UniProt Swiss‐Prot knowledgebase, and on the basis of in silico analysis have divided them into I1 and I2 superfamilies. The sequence analysis has provided a framework for in silico annotation enabling us to carry out computer‐based functional characterization of the UniProtKB/TrEMBL entry Q59AA4 from Conus miles and to predict it as a member of the I2 superfamily. Furthermore, we have predicted the mature toxin of this entry and have proposed that it may be an inhibitor of voltage‐gated potassium channels. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd.