Many cell surface receptors activate phosphoinositidase(s) C, via G proteins that catalyze the hydrolysis of phosphatidylinositol 4,5-biphosphate to produce the second messengers, inositol(1,4,5)trisphosphate [Ins(1,4,5)P₃] and diacylglycerol (1). Ins(1,4,5)P₃ interacts with specific receptor populations of ligand-gated channels to mobilize nonmitochondrial intracellular calcium (Ca²⁺) stores (1). Because Ins(1,4,5)P₃ is very hydrophilic, it cannot readily cross the intact plasma membrane. Consequently, Ins(1,4,5)P₃-induced Ca²⁺ release was initially demonstrated in permeabilized pancreatic acinar cells (2), and all subsequent studies in cells have involved the introduction of Ins(1,4,5)P₃ by rendering a cell population permeable (3), using microinjection techniques (4) or by the presentation of chemically modified membrane-permeable Ins(1,4,5)P₃ analogs, such as photolabile “caged Ins(1,4,5)P₃” (5). An alternative approach involves disruption of the plasma membrane and preparation of microsomes from the intracellular vesicular Ca²⁺ stores (6,7), however, these preparations exhibit a loss of Ins(1,4,5)P₃ responsiveness compared to cells. The author will describe a ⁴⁵Ca²⁺-release assay used to monitor Ins(1,4,5)P₃-induced Ca²⁺ mobilization from nonmitochondrial intracellular Ca²⁺ stores using “cytosol-like” buffer (CLB) and permeabilized SH-SY5Y neuroblastoma cell populations.