This article describes the construction, testing, calibration and application of ion-selective double-barrelled microelectrodes based on neutral carrier or ion-exchanger liquid membranes. At present the use of ion-selective microelectrodes is the only method available for the direct and continuous measurement of ion activities in the intra-and extracellular spaces, on membrane surfaces, and even in subcellular organelles of a variety of intact cells. Intracellular ion activities in identified neurones of the central nervous system of the leech (Hirudo medicinalis L.) have been measured with K+-, Na+-, H+-, Mg2+- and Ca2+-selective double-barrelled microelectrodes under various experimental conditions, such as changes in ion composition of the external medium, application of neurotransmitters and exposure to drugs. This approach has provided significant information about electrochemical potentials of ions, properties of receptor-coupled ion channels, and membrane transport related to ion activities in this nervous system.