Nanosecond pulsed electric fields (nsPEFs) present a novel and effective method for cancer ablation by eradicating the ubiquitous cancer hallmark of apoptosis evasion and enforcing cancer programmed cell death. To develop nsPEFs as an anticancer method, a comprehensive understanding of cell death mechanisms is required. The overall objective of this dissertation is to elucidate molecular mechanisms underlying effects of nsPEFs on E4 murine squamous cell carcinoma and human T-cell Jurkat clones that are wildtype, deficient in FADD (ΔFADD) and deficient in caspase-8 (ACas-8). The overall hypothesis is that nsPEFs eliminate cancer cells through activating caspase-dependent and caspase-independent programmed cell death pathways that originate from plasma membranes and/or intracellular membranes, especially mitochondria and endoplasmic reticulum. The specific aims are to: 1) determine whether nsPEFs induce cell death in E4 cells and Jurkat clones through caspase activation, 2) determine involvement of caspase-independent mechanisms in nsPEF-induced apoptosis in E4 cells and Jurkat clones and 3) determine signaling pathways for caspase-dependent apoptosis mediated by nsPEFs in E4 cells and Jurkat clones in vitro. Our findings are: 1) nsPEFs appear to induce apoptosis through the extrinsic pathway in E4 cells because caspase activation occurs before cytochrome c release. However, results from Jurkat clones, which have mutations in the extrinsic apoptosis pathway, suggest that apoptosis is induced by nsPEFs through the intrinsic pathway. 2) NsPEFs have rapid effects to decrease the mitochondria membrane potential and in Jurkat cells this occurs in the presence or absence of calcium or sodium. 3) NsPEFs activate calcium-dependent calpain pathways in E4 cells, which, in part, contribute to Bid cleavage, with calcium contributions from extracellular (70%) and intracellular (30%) compartments and 4) Cells respond to nsPEFs differently in terms of caspase-dependent apoptotic pathways depending on cell type. Apoptosis is induced through the intrinsic pathway in Jurkat cells, whereas in E4 cells, both intrinsic and extrinsic pathways are activated upon nsPEF-stimulation. These findings provide new insights into mechanisms by which nsPEFs promote cancer cell death, and demonstrate that nsPEFs administration can be utilized as a new therapeutic strategy for cancer ablation.