We have found that the hemolytic and cytotoxic activities of myristoylated Nef N-terminal peptides require a net positive charge in the first seven amino residues of the sequence. The activities are considerably less dependent on the secondary structure of the peptides. Film balance studies showed that both active and inactive peptides interacted with neutral phospholipid monolayers, suggesting that binding to neutral lipids was not a sufficient condition for lytic activity. It was also found that nonmyristoylated N-terminal peptide did not interact to the same extent with the monolayer, indicating that myristoylation was essential for lipid interaction. It is considered that the positively charged residues of the proximate N terminus of Nef interact with acidic lipids of biological membranes, reinforcing the weak membrane-targeting properties of the myristyl chain. Parallels are drawn between this mode of interaction with membranes and that of members of the Src family of proteins, which are also myristoylated and have positively charged residues in their proximate N termini. In particular, these proteins and Nef also have serine residues in their proximal N-terminal regions, which when phosphorylated could neutralize the positive charge and thus provide a mechanism for modulating membrane interaction.