Neuropeptides are inter-cellular signaling molecules occurring throughout animals. Most neuropeptides bind and activate G-protein-coupled receptors, but some also activate ionotropic receptors (or "ligand-gated ion channels"). This is exemplified by the tetra-peptide H-Phe-Met-Arg-Phe-NH(2) (FMRFamide (FMRFa)), which activates mollusk and annelid FMRFa-gated sodium channels (FaNaCs) from the trimeric degenerin/epithelial sodium channel superfamily. Here, we explored the structure-activity relationships determining FMRFa potency at mollusk and annelid FaNaCs in the light of emerging structural data, using synthetic neuropeptide analogs, heterologous expression, and two-electrode voltage clamp. Substitutions of the FMRFa N-terminal phenylalanine residue (F1) and methionine residue (M2) decreased or abolished FMRFa potency at mollusk Aplysia kurodai FaNaC but had little effect at annelid Malacoceros fuliginosus FaNaC1. Conversely, F4 substitutions had little effect on FMRFa potency at A. kurodai FaNaC but either abolished, strongly decreased, or slightly increased potency at M. fuliginosus FaNaC1. Accordingly, recently published high-resolution FaNaC structures show that F1 and F4 residues orient deep into the neuropeptide-binding pockets of A. kurodai FaNaC and M. fuliginosus FaNaC1, respectively. We also use noncanonical amino acid substitutions in A. kurodai FaNaC to describe the physico-chemical determinants of FMRFa F1 binding to A. kurodai FaNaC aromatic side chains. Our results show that the "deeper" of the two FMRFa phenylalanine residues in the binding pocket is crucial for FMRFa potency despite the peptide orienting very differently into the homologous binding sites of two closely related receptors.