Stomata play a crucial role in the regulation of gas exchange through the pore between a pair of guard cells. Recently, we have reported a gene named STOMAGEN shows unnaturally increased stomata in the over-expressed lines. The 45-amino-acid cysteine-rich peptide at the C-terminus of the STOMAGEN precursor act as a hormonally active peptide which contains three intermolecular disulfide bonds. STOMAGEN is a member of Epidermal Patterning Factor (EPF) family. EPF1 and EPF2 are also in the family, which lead to the contrary phenotype with decreased stomatal formation when overexpressed in Arabidopsis. However, the essential region of EPF1 and EPF2 is not clear. In this presentation, we produced the bioactive peptides derived from EPF1 and EPF2. We synthesized these peptides using Fmoc chemistry, or obtained recombinant peptides using E. coli, and then refolded these peptides. Folded EPF1 and EPF2 peptides significantly decreased stomatal density in the abaxial leaf epidermis at the concentrations around 10 μM. We analysed the arrangement of the intermolecular disulfide bonds in EPF1 by enzymatic digestion of this peptide, and revealed that the fifth and sixth cysteine residues that are additional, not conserved in the family are bound each other through a disulfide bond. These results suggest that the C-terminal cysteine-rich region of EPF family is sufficient to function as bioactive peptides, and share common backbone fixed by the conserved cysteine residues.