Continuous perfusion of immobilized electropermeabilized SH-SY5Y neuroblastoma cells was utilised as a novel approach to the assessment of incremental activation and inactivation of myo-inositol 1,4,5-trisphosphate (IP3)-induced calcium (Ca2+) mobilisation (IICM). SH-SY5Y cells when stimulated with sub-optimal IP3 exhibited a rapid concentration dependent activation of Ca2+ mobilization followed by a partial inactivation. Although this partial inactivation allowed net Ca2+ mobilized to be stringently returned to basal levels, a concentration-dependent depletion of the store was maintained while ever perfusion with the stimulating IP3 concentration was sustained. This partial inactivation of IP3-induced quantal Ca2+ release (QCR) was only compromised if cells, with replete Ca2+ stores, were perfused with supra-maximally effective concentrations of IP3 (5-10 microM). Thus, at supra-optimal IP3 concentrations, a reproducible plateau of Ca2+ release lying 50-150 nM above the basal Ca2+ concentration was observed. Feedback on IP3R sensitivity by gross cytosolic Ca2+ levels could be eliminated as the sustained and exclusive mediator of incremental activation/inactivation cycle of IICM in SH-SY5Y cells, since released Ca2+ was perfused away from the immobilized cells. Thus, while ever the cells were continuously perfused with IP3, impressive incremental inactivation was apparent. Additionally, IP3R partial agonists were found to exhibit lower intrinsic activity for both activation and inactivation of QCR, suggesting that ligand-induced inactivation of the IP3R was more important than inactivation mechanisms reliant on either Ca2+ flux through the channel and/or calcium store depletion. Therefore, we suggest that, in perfused SH-SY5Y cells, the most parsimonious explanation of our data is that IP3 binding probably activates and then partially inactivates its receptor in a concentration-dependent fashion to produce the QCR phenomenon.