Calcicludine, a 60-amino acid protein isolated from the green mamba venom, has been recently identified as blocking a large set (i.e., L-, N-and P-type) of Ca 2؉ channels. The threedimensional structure of calcicludine has been determined by NMR and molecular modeling using a data set of 723 unambiguous and 265 ambiguous distance restraints, as 33 ⌽ and 13 1 dihedral angle restraints. Analysis of the 15 final structures (backbone root-mean-square deviation ؍ 0.6 A ˚) shows that calcicludine adopts the Kunitz-type protease inhibitor fold. Its three-dimensional structure is similar to that of snake K ؉ channel blockers dendrotoxins. Conformational differences with protease inhibitors and dendrotoxins are localized in the 3 10 helix and loop 1 (segments 1-7 and 10-19), the extremity of the -hairpin (segment 27-30), and loop 2 (segment 39-44). These regions correspond to the functional sites of bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxins. The positioning of the N-terminal segment 1-7 relative to the rest of the protein is characteristic of calcicludine. The involvement of this segment and the positively charged K31 at the tip of the -hairpin in the biological activity of calcicludine is discussed.