Intracellular activities of K, Na, H, Cl, and Ca ions are kept at an optimal level for maintenance of normal cellular functions. Furthermore, many cellular functions are regulated directly or indirectly by these ion activities in cells. The use of ion-selective microelectrodes has emerged as the only method for measuring directly and continuously intracellular ion activities of intact cells. In recent years, they have been extensively used for measurements of intracellular ion activities in cardiac muscle and other tissues. This article concerns both intracellular ion activities of cardiac muscle and intracellular application of ion-selective microelectrodes. Intracellular ion activities in cardiac muscle cells have been measured under several experimental conditions, such as changes in ion composition of bathing fluid and exposure to certain drugs. This approach has provided significant information about electrochemical potentials of ions, membrane transport, and cellular functions related to ion activities. Relevant data and information is presented for K+, Na+, H+, Cl-, and Ca2+. Although ion-selective microelectrodes are widely used, this technique has certain assumptions, problems, and limitations that vary with the types of microelectrodes. The appendix of this article reviews basic principles for intracellular application of ion-selective microelectrodes and examines pertinent assumptions, problems, and issues. Particular features of K+-, Na+-, H+-, Cl--, and Ca2+-selective microelectrodes are described.