The Arabidopsis thaliana genome contains five genes that encode two pore K+ (TPK) channels. The most abundantly expressed isoform of this family, TPK1, is expressed at the tonoplast where it mediates K+ -selective currents between cytoplasmic and vacuolar compartments. TPK1 open probability depends on both cytoplasmic Ca2+ and cytoplasmic pH but not on the tonoplast membrane voltage. The channel shows intrinsic rectification and can be blocked by Ba2+, tetraethylammonium, and quinine. TPK1 current was found in all shoot cell types and shows all of the hallmarks of the previously described vacuolar K (VK) tonoplast channel characterized in guard cells. Characterization of TPK1 loss-of-function mutants and TPK1-overexpressing plants shows that TPK1 has a role in intracellular K+ homeostasis affecting seedling growth at high and low ambient K+ levels. In stomata, TPK1 function is consistent with vacuolar K+ release, and removal of this channel leads to slower stomatal closure kinetics. During germination, TPK1 contributes to the radicle development through vacuolar K+ deposition to provide expansion growth or in the redistribution of essential minerals.