Many cell-surface receptors via G-proteins activate phosphoinositidase C, which catalyzes the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce the second messengers, myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3 or IP3 ] and diacylglycerol (1 ). Ins(1,4,5)P3 interacts with a specific receptor populations of ligand-gated channels to mobilize nonmitochondrial intracellular Ca2+ stores (1 ). Because Ins(1,4,5)P3 is plasma-membrane-impermeant, this phenomenon was first demonstrated in permeabilized pancreatic acinar cells (2 ), and all subsequent studies in cells have involved 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(l,4,5)P3 analogs, such as photolabile “caged-IP3 ” (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. Here we describe a 45 Ca45 -release assay and a fluo-3 assay, two methods we use to monitor Ins(l,4,5)P3 -induced Ca2+ mobilization from nonmitochondrial intracellular Ca2+ stores using cytosol-like buffer (CLB) and permeabilized SH-SY5Y neuroblastoma cell populations. The 45 Ca2+ -release assay was similar to those previously described (3 , 8 ), and involves preloading a “spike” of 45 Ca2+ into the intracellular Ca2+ stores of permeabilized cells and then monitoring the resultant release of 45 Ca2+ induced by concentrations of Ins(1,4,5)P3 and other agonists. We have utilized this assay to assess the intrinsic activity of Ins(1,4,5)P3 a wide range of Ins(l,4,5)P3 analogs, and other Ca2+ -mobilizing agents. The assay can be undertaken at a low cell density to minimize cellular metabolism of the inositol polyphosphates, or at high cell density to allow metabolic time-courses to be associated with Ca2+ mobilization.