We have studied the responses of iron regulatory protein-1 (IRP-1) to extra- and intracellular sources of reactive oxygen intermediates (ROIs). IRP-1 is a cytoplasmic RNA-binding protein that regulates iron metabolism following its activation by iron deficiency, nitric oxide, and administration of H2O2 or antimycin A, an inhibitor of the respiratory chain (Hentze, M. W., and Kühn, L. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8175-8182). We show that 10 microM H2O2 suffice for complete IRP-1 activation within 60 min when H2O2 is generated extracellularly at steady-state. By contrast, rapid cellular H2O2 degradation necessitates a 5-10-fold higher bolus dose. To study IRP-1 responses to intracellular oxidative stress, mitochondrial respiration was inhibited with antimycin A (to generate oxidative stress by leakage of ROIs from complex III), or catalase was blocked with 3-amino-1,2,4-triazole (to diminish H2O2 degradation); in parallel, 2',7'-dichlorodihydrofluorescein diacetate was used as a redox-sensitive probe to monitor intracellular H2O2 levels by fluorescence-activated cell sorting. Catalase inhibition elevates intracellular H2O2, but surprisingly does not cause concomitant IRP-1 activation. Following antimycin A treatment, IRP-1 is activated, but the activation kinetics lag behind the rapid increase in detectable intracellular H2O2. IRP-1 is thus activated both by extra- and intracellular generation of ROIs. While extracellular H2O2 rapidly activates IRP-1 even without detectable increases in intracellular H2O2, intracellular H2O2 elevation is not sufficient for IRP-1 activation. IRP-1 thus represents a novel example of an H2O2-regulated protein that responds differentially to alterations of extra- and intracellular H2O2 levels. Our data also suggest that a direct attack on the 4Fe-4S cluster of IRP-1 by H2O2 (or an H2O2-derived reactive species) represents an unlikely explanation for IRP-1 activation by oxidative stress.