Progress in the study of Nox protein expression has been impeded because of the paucity of immunological probes. The large subunit of human phagocyte flavocytochrome b558 (Cytb), gp91phox, is also the prototype member of the recently discovered family of NADPH oxidase (Nox) proteins. In this study, we have evaluated the use of two anti-gp91 phox monoclonal antibodies, 54.1 and CL5, as immunoprobes for Nox family proteins. Sequence alignment of gp91phox with Nox1, Nox3 and Nox4 identified regions of the Nox proteins that correspond to the gp91 phox epitopes recognized by mAb 54.1 and CL5. Antibody 54.1 produced positive immunoblots of recombinant C-terminal fragments of these homologous proteins expressed in E. coli. Furthermore, only mAb 54.1 recognized full-length murine and human Nox3 expressed in HEK-293 cells, in immunoblots of alkali-stripped or detergent-solubilized membranes. 54.1 recognized Nox3 expression-specific proteins with Mr 30,000, 50,000, 65,000 and 88,000 for the murine protein and Mr of 38,000-58,000, 90,000, 100,000-130,000 and a broad species of higher than 160,000 for the human protein. We conclude that mAb 54.1 can serve as a probe of Nox3 and possibly other Nox proteins, if precautions are taken to remove GRP 58 and other crossreactive membrane-associated or detergent-insoluble proteins from the sample to be probed. © 2005 Elsevier B.V. All rights reserved.