Dual oxidases (Duox1 and Duox2) are plasma membrane-targeted hydrogen peroxide generators that support extracellular hemoperoxidases. Duox activator 2 (Duoxa2), initially described as an endoplasmic reticulum resident protein, functions as a maturation factor needed to deliver active Duox2 to the cell surface. However, less is known about the Duox1/Duoxa1 homologues. We identified four alternatively spliced Duoxa1 variants and explored their roles in Duox subcellular targeting and reconstitution. Duox1 and Duox2 are functionally rescued by Duoxa2 or the Duoxa1 variants that contain the third coding exon. All active maturation factors are cotransported to the cell surface when coexpressed with either Duox1 or Duox2, consistent with detection of endogenous Duoxa1 on apical plasma membranes of the airway epithelium. In contrast, the Duoxa proteins are retained in the endoplasmic reticulum when expressed without Duox. Duox1/Duoxa1α and Duox2/Duoxa2 pairs produce the highest levels of hydrogen peroxide, as they undergo Golgi-based carbohydrate modifications and form stable cell surface complexes. Cross-functioning pairs that do not form stable complexes produce less hydrogen peroxide and leak superoxide. These findings suggest Duox activators not only promote Duox maturation, but they function as part of the hydrogen peroxide-generating enzyme.—Morand, S., Ueyama, T., Tsujibe, S., Saito, N., Korzeniowska, A., Leto, TL Duox maturation factors form cell surface complexes with Duox affecting the specificity of reactive oxygen species generation.