Iron regulatory proteins 1 and 2 (IRP-1, IRP-2) interact with iron-responsive elements (IREs) present in the 5'- or 3'-untranslated regions (UTR) of several mRNAs coding for proteins in iron metabolism. Whereas binding of IRP-1 and -2 to an IRE in the 5'-UTR inhibits mRNA translation in vitro, it has remained unknown whether either endogenous protein is sufficient to control translation in mammalian cells. We analyzed this question by taking advantage of published mutant IREs that are exclusively recognized by either IRP-1 or IRP-2 in vitro. These IREs were inserted into the 5'-UTR of a human growth hormone reporter mRNA, and translational regulation was measured in stably transfected mouse L cells. Cells cultured in iron-rich or -depleted medium were labeled with [35S]methionine, and secreted growth hormone was immunoprecipitated. IREs with loop sequence specific for IRP-1 (UAGUAC), IRP-2 (CCGAGC), or both proteins (GAGUCG and the wild-type CAGUGC sequence) all mediated translational regulation, in contrast to a control sequence (GCUCCG) that binds neither IRP-1 nor IRP-2. Control experiments excluded IRP-1 binding to the IRP-2-specific sequence in vivo. The present data demonstrate that IRP-1 and IRP-2 can independently function as translational repressors in living cells.