Ca²⁺ is an important ion that controls almost every function in a cell. Activator Ca²⁺ can be released from intracellular Ca²⁺ stores, and there are various ways to study this release. Here, we introduce a technique that uses radioactive ⁴⁵Ca²⁺ to quantitatively measure the unidirectional release of Ca²⁺ from the nonmitochondrial Ca²⁺ stores in monolayers of cultured cells. This technique can be used in cells with an intact plasma membrane as well as in cells in which the plasma membrane has been permeabilized.