Iron regulatory proteins (IRP)-1 and 2 are cytoplasmic mRNA-binding proteins that control intracellular iron homeostasis by regulating the translation of ferritin mRNA and stability of transferrin receptor mRNA in an iron-dependent fashion. Although structurally and functionally similar, the two IRP are different in their mode of regulation, pattern of tissue expression and modulation by multiple factors, such as bioradicals. In the present study RNA bandshift assays demonstrated that IRP-2, but not IRP-1, activity was higher in cultured cells than in tissues. Increased expression of IRP-2 in cell lines was not related to immortalization and differentiation but seemed associated to cell proliferation, although not closely dependent on cell growth rate. As a growing cell consumes more iron than its quiescent counterpart, we assessed the iron status of cell lines and found that ferritin content was lower than in tissues. Analysis of IRP activity in cell lines supplemented with heme or non-heme iron and in livers of iron-loaded and iron-deficient rats indicated that IRP-2 responds more promptly than IRP-1 to modulations of iron content. We propose that enhanced IRP-2 activity in cultured cells could be due to a proliferation-dependent, relative iron deficiency that is sensed first by IRP-2.