NOX1, an NADPH oxidase expressed predominantly in colon epithelium, shows a high degree of similarity to the phagocyte NADPH oxidase. However, superoxide generation by NOX1 has been difficult to demonstrate. Here we show that NOX1 generates superoxide when co-expressed with the p47(phox) and p67(phox) subunits of the phagocyte NADPH oxidase but not when expressed by itself. Since p47(phox) and p67(phox) are restricted mainly to myeloid cells, we searched for their homologues and identified two novel cDNAs. The mRNAs of both homologues were found predominantly in colon epithelium. Differences between the homologues and the phagocyte NADPH oxidase subunits included the lack of the autoinhibitory domain and the protein kinase C phosphorylation sites in the p47(phox) homologue as well as the absence of the first Src homology 3 domain and the presence of a hydrophobic stretch in the p67(phox) homologue. Co-expression of NOX1 with the two novel proteins led to stimulus-independent high level superoxide generation. Stimulus dependence of NOX1 was restored when p47(phox) was used to replace its homologue. In conclusion, NOX1 is a superoxide-generating enzyme that is activated by two novel proteins, which we propose to name NOXO1 (NOX organizer 1) and NOXA1 (NOX activator 1).