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Novel promoters that confer root-specific expression would be useful for engineering resistance against problems of nutrient and water absorption by roots. In this study, the reverse transcriptase polymerase chain reaction was used to identify seven genes with root-specific expression in rice. The isolation and characterization of upstream promoter regions of five selected genes rice root-specific promoter (rRSP) 1 to 5 (rRSP1–rRSP5) and A2P (the promoter of OsAct2) revealed that rRSP1, rRSP3, and rRSP5 are particularly important with respect to root-specific activities. Furthermore, rRSP1, rRSP3, and rRSP5 were observed to make different contributions to root activities in various species. These three promoters could be used for root-specific enhancement of target gene(s).