The thyroid plasma membrane contains a Ca(2+)-regulated NADPH-dependent H2O2-generating system which provides H2O2 for the thyroid-peroxidase-catalyzed biosynthesis of thyroid hormones. The molecular nature of the membrane-associated electron transport chain that generates H2O2 in the thyroid is unknown, but recent observations indicate that a flavoprotein containing a FAD prosthetic group is involved. Solubilization was reinvestigated using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), Triton X-100, and high salt concentrations. Chaps eliminated about 30% of the proteins, which included a ferricyanide reductase, without affecting the H2O2-generating system. Similarly, Triton X-100 alone did not extract the NADPH oxidase. An NADPH-oxidase activity, which was measured in the presence of the artificial electron acceptor potassium ferricyanide, was solubilized by increasing the ionic strength to 2 M KCl. This NADPH-ferricyanide reductase activity was shown to belong to the H2O2-generating system, although it did not produce H2O2. It was still Ca2+ dependent and H2O2 production was restored by decreasing the ionic strength by overnight dialysis. No H2O2 production activity was detected after sucrose density gradient centrifugation of the dialyzed solubilized enzyme, but a well-defined peak of NADPH oxidation activity with a sedimentation coefficient of 3.71 S was found in the presence of K3Fe(CN)6. These results suggest that some unknown component(s) (phospholipid or protein) is removed during sucrose density gradient centrifugation. Finally, thyrotropin, which induces NADPH oxidase and regulates H2O2 production in porcine thyrocytes in primary culture, also induced the NADPH-K3Fe(CN)6 reductase activity associated with the H2O2-generating system. Thus, this enzyme seems to be another marker of thyroid differentiation.