Plasmatocyte Spreading Peptide (PSP) is a 23 amino acid cytokine that activates a class of insect immune cells called plasmatocytes. The 3-D structure of PSP consists of an unstructured N-terminus (residues 1-6) and a well-structured core (residues 7-23) stabilized by a disulfide bond and a short ß-hairpin. Within the structured core, previous mutagenesis studies identified key residues required for biological activity. These include the cysteines necessary for disulfide bond formation and charged residues within the b-hairpin. The unstructured N-terminus is also essential as deletion of the first six residues eliminates all spreading activity. Alanine substitution of the first three residues (Glu1-Asn2-Phe3) indicated that only F3A results in a loss of activity commensurate with the N-terminal deletion. In this study, we further characterized structural determinants of the N-terminus required for activity. Adding a hydroxyl group to the aromatic ring of Phe3 (making a Tyr) greatly reduced activity, whereas addition of a fluorine (p-fluoro) did not. Substitutions that changed the chirality or that replaced the aromatic ring of Phe3 with a branched aliphatic chain (making a Val) also greatly decreased activity. Addition of a methylene group to Val (making a Leu) partially restored activity while removal of a methylene group from Phe (phenyl-Gly) eliminated all activity. Together these results indicated that a branched carbon chain with a methylene spacer at the third residue is the minimal structural motif required for activity. To confirm the importance of Phe3 within the N-terminus, we also tested deletions of Glu1 and Asn2. While previous experiments showed that alanine replacement of Glu1 increased activity of PSP, we found here that deletion of Glu1 eliminated all activity. Additional experiments indicated that the charged N-terminal amine and backbone of Glu1 are key determinants required for PSP activity.