Stomatal development in plants is regulated by defensin-like secretory epidermal patterning factor (EPF) peptide hormones. Only one of these, stomagen, is a positive regulator, whereas EPF1, EPF2, and possibly others are negative regulators. Here we explore the structure-function relationships of EPFs, by integrating NMR and semi-in vitro stomagen experiments. We show that stomagen is composed of a loop and a scaffold containing three disulphide bonds. A mutant composed of the stomagen loop and the EPF2 scaffold positively regulates the stomatal density on Arabidopsis cotyledons. The reciprocal mutant composed of the EPF2 loop and the stomagen scaffold acts negatively. Deletion of the disulphide bond introduces unfolding and inactivity. Our results suggest that the loop confers the functional specificity of EPFs and that the scaffold is structurally required for their activity. This structural decomposition approach to elucidating the functional site could be adapted for the analysis of other cysteine-rich peptide families.