This protocol describes a technique to measure Ca²⁺ release from the nonmitochondrial intracellular Ca²⁺ stores in monolayers of saponin-permeabilized cells cultured in 12-well 4-cm² clusters. The ⁴⁵Ca²⁺-flux technique described here can only be applied to cell types that still adhere to the plastic after exposing them to saponin. We describe the permeabilization procedure, the loading of the nonmitochondrial Ca²⁺ stores with ⁴⁵Ca²⁺, and the subsequent ⁴⁵Ca²⁺ efflux.