PREMISE OF THE STUDY: The cyclotide kalata B1 is found in the leaves of Oldenlandia affinis and is a potent insecticidal and nematocidal molecule. This peptide is cleaved from a precursor protein, Oak1, and ligation of the N- and C-termini occurs to form a continuous peptide backbone. The subcellular location of the excision and cyclization reactions is unknown, and there is debate as to which enzyme catalyzes the event. To determine where in the plant cell Oak1 is processed, we prepared constructs encoding GFP (green fluorescent protein) linked to the cyclotide precursor Oak1. METHODS: The GFP constructs were transiently expressed in the leaves of Nicotiana benthamiana, and GFP fluorescence was observed in living cells using confocal microscopy. A Fei Mao (FM) styryl dye was infiltrated into whole leaves that were still growing and expressing GFP constructs, enabling the plasma membrane and the tonoplast to be highlighted for visualization of the vacuole in living cells. KEY RESULTS: The full length Oak1 precursor directed GFP to the vacuole, suggesting that excision and cyclization of the cyclotide domain occurs in the vacuole where the cyclotides are then stored. The N-terminal propeptide and N-terminal repeat of Oak1 were both sufficient to target GFP to the vacuole, although the C-terminal propeptide, which is essential for cyclization, was not a targeting signal. CONCLUSIONS: The vacuolar location of cyclotides supports our hypothesis that the vacuolar processing enzyme, asparaginyl endoproteinase, has a pivotal role in excision and cyclization from cyclotide precursors.