The superoxide (O2-)-generating NADPH oxidase of human neutrophils consists of membrane-bound and cytosolic proteins that assemble in the plasma membrane of activated cells. To date, most of our understanding of the assembly of the NADPH oxidase has been obtained through the use of a cell-free assay, and a number of peptides that mimic regions of NADPH oxidase proteins have been shown to block oxidase assembly using this assay. However, the cell-free assay provides an incomplete representation of the assembly and regulation of the NADPH oxidase in vivo, and it has become necessary to develop methods for introducing biomolecules, such as peptides, into intact neutrophils where their effects can be investigated. One such method is electropermeabilization. Although this method has been used previously with human neutrophils, it has not been well characterized. We report here a detailed characterization of the electropermeabilized neutrophil assay system, including optimal conditions for membrane electropermeabilization with maximal retention of functional capacity, optimal conditions for analyzing the effects of experimental peptides, quantification of internalized peptide concentration, and molecular size limits for diffusion of molecules into these cells. Our results demonstrate that optimal neutrophil permeabilization (98-100%) can be achieved using significantly lower electrical fields than previously reported, resulting in the retention of higher levels of O2(-)-generating activity. We also found that biomolecules as large as 2.3 kDa readily diffuse into permeabilized cells. Analysis of flavocytochrome b peptides that were shown previously to inhibit NADPH oxidase activity in a cell-free assay demonstrated that these peptides also blocked O2- production in electropermeabilized human neutrophils; although at higher effective concentrations than in the cell-free system. Thus, electropermeabilized neutrophils provide a model system for evaluating the effects of peptides and other pharmacological agents in intact cells which closely mimic neutrophils in vivo.