Iron regulatory proteins (IRPs)-1 and -2 bind specific mRNA hairpin structures known as iron-responsive elements and thereby post-transcriptionally regulate proteins involved in iron uptake, storage, and utilization. In this study, we compared modulation of the RNA-binding activities of IRP-1 and IRP-2. We show that in vitro RNA-binding can be inhibited for each IRP by the alkylation of free sulfhydryl groups with N-ethylmaleimide, or by oxidation with diamide. The in vivo iron regulation of IRP-1 and IRP-2 appeared to involve different pathways. Both proteins are activated in Ltk- cells following iron chelation. This induction, however, was distinguishable by the addition of translation inhibitors, which temporarily delayed activation of IRP-1 by up to 8 h, but fully blocked IRP-2 induction for up to 20 h. The activation of IRP-2 was also prevented by transcription inhibition with actinomycin D. Further analysis revealed that, while both IRPs are rapidly inactivated following iron treatment of iron-depleted cells, the repression of IRP-2 was again completely translation dependent. Immunoblot analysis suggests that iron modulation of IRP-1 activity is predominantly a posttranslational process. This contrasts with IRP-2, whose activation reflected the accumulation of stable IRP-2 protein by de novo synthesis. IRP-2 inactivation/degradation occurred upon readdition of iron, but it required translation of another protein. The existence of an independent regulator of IRP-2 may help explain the differential regulation and expression of the two IRP proteins in different tissues and cell lines.