Melittin is the principal protein component of bee venom and is thought to function as a lytic agent. Despite its predominantly hydrophobic character, melittin is soluble as a tetramer in aqueous salt solutions. We report here on the determination of the crystal structure of tetrameric melittin at 2.8-A resolution by the method of multiple isomorphous replacement, followed by partial atomic refinement at 2.0-A resolution. The melittin tetramer contains a noncrystallographic 2-fold axis of symmetry in addition to a crystallographic 2-fold axis, so that the four polypeptide chains have nearly identical structures. The noncrystallographic 2-fold axis was utilized twice during the determination of the structure. The multiple isomorphous replacement electron density map was averaged over this 2-fold axis before model building and strict noncrystallographic symmetry was assumed during the initial stages of atomic refinement. The 2.8-A resolution electron density map suggests that the melittin monomer contains two alpha-helical regions separated by a non-alpha-helical segment at residues 11 and 12. Difference maps at 2.0-A resolution tend to confirm this structure and reveal that at least six solvent molecules are bound to the melittin tetramer in the crystal. The relatively high occupancies of four of these suggest that they are ions of crystallization rather than water molecules.