Polymorphonuclear neutrophils (PMN) have a remarkable capacity for generation of large amounts of reactive oxygen species in response to a variety of infectious or inflammatory stimuli, a process known as the respiratory burst that involves activation of a multicomponent NADPH oxidase. Given their short life span, PMN are not amenable to most molecular biology methods for studying activation of this oxidant-generating system. We have explored a variety of methods for introduction of components of the phagocytic oxidase (phox system) into the promyelocytic erythroleukemia cell line, K-562. Here, we describe a series of cloned K-562 cell lines that were retrovirally transduced for stable production of one or more essential components of the phagocytic oxidase (phox) complex. We outline methods for the use of these transfectable cells for investigating structure, function, and signaling requirements for assembly and activation of the phox system. These versatile lines can be used to examine effects of genetic polymorphisms or mutations in phox components associated with chronic granulomatous disease, to serve as a system for testing gene therapy vectors designed to correct the defective oxidase, to study cross-functioning with recently described phox component homologs, or to explore signaling components involved in regulation of the respiratory burst.