We have investigated the effects on the hemolytic activity of the 26 individual amino acids making up melittin's sequence using a complete set of synthetic leucine substitution analogs. The relative dose to cause 50% hemolysis (HD50) for each analog was determined. Lysine-7 and tryptophan-19 were found to play an essential role as evidenced by the dramatic decrease in hemolytic activity found for their leucine substitution analogs relative to melittin. In contrast, extension of melittin's potential alpha-helical array by substitution of the residues making up the" hinge" region, and increase of its overall amphipathicity by substitution of proline-14 or by the individual substitution of three of the C-terminal basic residues, yielded significant reproducible increases in hemolytic activity.