The AP2/EREBP transcription factors play important roles in plant development and in the responses of plants to biotic and abiotic stresses. All members of the EREBP subfamily described to date are from dicotyledonous plants. In this paper, we describe the cloning and characterization of a rice gene, OsEBP-89, encoding a protein 326 amino acids long with a typical EREBP domain; this is the first report of an EREBP transcription factor in a monocotyledonous plant. Except for the EREBP domain, the OsEBP-89 protein does not have substantial sequence similarities to other members of the subfamily. The DNA-binding activity of the EREBP domain was confirmed by electrophoretic mobility-shift assays. An activation domain rich in acidic amino acids was identified by using a yeast one-hybrid system. Two putative nuclear-localization signals were also identified. The results of northern blot hybridization experiments showed that the transcript of the OsEBP-89 gene accumulates primarily in immature seeds, roots, and leaves (low levels). More detailed information about the pattern of OsEBP-89 gene expression was obtained by histochemical studies of transgenic rice plants carrying an OsEBP-89 5'/GUS reporter gene. The reporter gene was expressed in the endosperm starting at 7 days after pollination and in the intercalary meristem of plants. Expression of OsEBP-89 was induced in roots of rice seedlings by treatment with ACC, NaCl, or 2,4-D. Two cis-acting elements, an endosperm motif and a primary PERE, are present upstream of the OsEBP-89 coding region and may be involved in regulating its expression. Collectively, these results suggest that the OsEBP-89 gene is a new member of the EREBP subfamily and may be involved in ethylene-dependent seed maturation and shoot development of rice.