The persistent influx of ions through nanopores created upon cellular exposure to nanosecond pulse electric fields (nsPEF) could be used to modulate neuronal function. One ion, calcium (Ca 2+ ), is important to action potential firing and regulates many ion channels. However, uncontrolled hyper-excitability of neurons leads to Ca 2+ overload and neurodegeneration. Thus, to prevent unintended consequences of nsPEF-induced neural stimulation, knowledge of optimum exposure parameters is required. We determined the relationship between nsPEF exposure parameters (pulse width and amplitude) and nanopore formation in two cell types: rodent neuroblastoma (NG108) and mouse primary hippocampal neurons (PHN). We identified thresholds for nanoporation using Annexin V and FM1-43, to detect changes in membrane asymmetry, and through Ca 2+ influx using Calcium Green. The ED50 for a single 600 ns pulse, necessary to cause uptake of extracellular Ca 2+ , was 1.76 kV/cm for NG108 and 0.84 kV/cm for PHN. At 16.2 kV/cm , the ED50 for pulse width was 95 ns for both cell lines. Cadmium, a nonspecific Ca 2+ channel blocker, failed to prevent Ca 2+ uptake suggesting that observed influx is likely due to nanoporation. These data demonstrate that moderate amplitude single nsPEF exposures result in rapid Ca 2+ influx that may be capable of controllably modulating neurological function.