To clarify the molecular mechanism that regulates iron (Fe) acquisition in graminaceous plants, a time-course analysis of gene expression during Fe deficiency stress was conducted using a rice 22K oligo-DNA microarray. Twenty-one genes for proteins that function in gene regulation were induced by Fe deficiency. Of these genes, a putative basic helix-loop-helix (bHLH) transcription factor gene, named OsIRO2, was strongly expressed in both roots and shoots during Fe deficiency stress. The expression of OsIRO2 was induced exclusively by Fe deficiency, and not by deficiencies in other metals. Expression of the barley HvIRO2 gene, which is a homologue of OsIRO2, was also induced by Fe deficiency. An in silico search revealed that IRO2 is highly conserved among graminaceous plants, which include wheat, sorghum, and maize. The cyclic amplification and selection of targets (CASTing) technique revealed that OsIRO2 bound preferentially to the sequence 5'-ACCACGTGGTTTT-3', and the electrophoretic mobility shift assay revealed 5'-CACGTGG-3' as the core sequence for OsIRO2 binding. Sequences similar to the OsIRO2-binding sequence were found upstream of several genes that are involved in Fe acquisition, such as OsNAS1, OsNAS3, OsIRT1, OsFDH, OsAPT1, and IDS3. The core sequence of the OsIRO2-binding sequence occurred more frequently in the upstream regions of Fe deficiency-inducible genes than in the corresponding regions of non-inducible genes. These results suggest that IRO2 is involved in the regulation of gene expression under Fe-deficient conditions.