The action of Ca2+ on H2O2 formation catalyzed by an NADPH-dependent H2O2-generating system was studied in a thyroid particulate fraction. It was shown that H2O2 formation was inhibited by EGTA and restored in the presence of micromolar concentrations of free Ca2+, and that Mg2+ was unable to restore the activity of the EGTA-inhibited particulate fraction. Consequently, NADPH-dependent H2O2 formation requires Ca2+, and the Ca2+ stimulation of this process is specific and reversible.