The conductance of the vacuolar membrane at elevated cytosolic Ca2+ levels is dominated by the slow activating cation selective (SV) channel. At physiological, submicromolar Ca2+ concentrations the SV currents are very small. Only recently has the role of 14-3-3 proteins in the regulation of voltage-gated and Ca2+-activated plasma membrane ion channels been investigated in Drosophila, Xenopus and plants. Here we report the first evidence that plant 14-3-3 proteins are involved in the down-regulation of ion channels in the vacuolar membrane as well. Using the patch-clamp technique we have demonstrated that 14-3-3 protein drastically reduces the current carried by SV channels. The current decline amounted to 80% and half-maximal reduction was reached within 5 s after 14-3-3-addition to the bath. The voltage sensitivity of the channel was not affected by 14-3-3. A coordinating role for 14-3-3 proteins in the regulation of plasma membrane and tonoplast ion transporters is discussed.