Mitochondria are crucial not only in energy metabolism but also in regulation of cell senescence and apoptosis. The strict control of inner mitochondrial membrane permeability and selective ion transport is essential for mitochondria functioning. Potassium ions homeostasis is an important process for mitochondrial optimal functioning. Potassium channels such as ATP-regulated, large conductance calcium activated and voltage dependent channels were observed in inner mitochondrial membrane in various mammalian tissues. Recently, we have identified potassium channels in inner mitochondrial membrane of potato Solanum tuberosum and Acanthamoeba castellanii. Currently we characterize mitochondrial potassium channels from one of Dictyostelium species. It is commonly used as a model organism to study cell differentiation, metabolism and programmed cell death. Preliminary experiments are focused on biophysical and pharmacological characterization of mitochondrial ion channels. Purified inner mitochondrial membranes (submitochondrial particles) were reconstituted into planar lipid bilayer. To form model membranes asolectin from soybean mixture of phospholipids was used. We observed two types of potassium selective ion channels in submitochondrial particle samples: a large- and small-conductance channels. Experiments were performed both in gradient solution 50/150 mM KCl (cis-trans) and in symmetrical solution 150/150 mM KCl at voltages from −50 to 50 mV. Regulation of the channel activity by divalent cations such as Ca2+ and Mg2+ was explored. Additionally, interaction of the ATP with mitochondrial potassium channels was characterized. The knowledge on mitochondrial ion channels may contribute to understanding molecular mechanism of Dictyostelium discoideum functioning.Thiswork was supportedby Polish Mitochondrial Network. MitoNet.pl