Expression of α-amylase genes in cereals is induced by both gibberellin (GA) and sugar starvation. In a transient expression assay, a 105-bp sugar response sequence (SRS) in the promoter of a sugar starvation highly inducible rice α-amylase gene, αAmy3, was shown previously to confer sugar response and to enhance the activity of the rice Act1 promoter in rice protoplasts. A 230-bp SRS-like sequence was also found in the promoter of another sugar starvation highly inducible rice α-amylase gene, αAmy8. The αAmy8 SRS contains a GA response sequence and was designated as αAmy8 SRS/GARS. In the present study, a transgenic approach was employed to characterize the function of the α-amylase gene SRSs in rice. We found that the αAmy3 SRS significantly enhances the endogenous expression pattern of the Act1 promoter in various rice tissues throughout their developmental stages. By contrast, the αAmy8 SRS/GARS significantly enhances Act1 promoter activity only in embryos and endosperms of germinating rice seeds. A minimal promoter fused to the αAmy8 SRS/GARS is specifically active in rice embryo and endosperm and is subject to sugar repression and GA induction in rice embryos. This sugar repression was found to override GA induction of αAmy8 SRS/GARS activity. Our study demonstrates that the α-amylase transcriptional enhancers contain cis-acting elements capable of enhancing endogenous expression patterns or activating sugar-sensitive, hormone-responsive, tissue-specific, and developmental stage-dependent expression of promoters in transgenic rice. These enhancers may facilitate the design of highly active and tightly regulated composite promoters for monocot transformation and gene expression. Our study also reveals the existence of cross-talk between the sugar and GA signaling pathways in cereals and provides a system for analyzing the underlying molecular mechanisms involved.