ABSTRACTThe crustacean hyperglycaemic hormone (CHH), a pleiotropic neuropeptide, belongs to a family of structurally related peptides, having six cysteine residues in conserved positions forming three disulphide bridges, and regulating several physiological processes in crustaceans and insects. Structure‐activity studies have shown that amidation of the C‐terminus is important to confer biological activity to CHH. In this study we investigated the function of the d‐Phe3 of the N‐terminal motif of Pontastacus leptodactylus CHH by a mutational analysis. The d‐Phe in position 3 was substituted by a d‐Ala and the functionality of the mutated analogue (Glp‐d‐A‐CHH) was tested by in vivo biological assays. The mutated analogue resulted far less active than its wild‐type counterparts, either in d‐ (Glp‐d‐CHH) or l‐ (Glp‐l‐CHH) configuration. These results suggest that Phe3 is essential for the biological activity of P. leptodactylus CHH, demonstrating that also the N‐terminus is involved in the binding with the receptor, and identifying in the Phe3 a hot spot for the peptide‐receptor binding. J. Exp. Zool. 323A: 121–127, 2015. © 2015 Wiley Periodicals, Inc.