Expression of α-amylase genes in both rice suspension cells and germinating embryos is repressed by sugars and the mechanism involves transcriptional regulation. The promoter of a rice α-amylase gene αAmy3 was analyzed by both loss- and gain-of-function studies and the major sugar response sequence (SRS) was located between 186 and 82 base pairs upstream of the transcription start site. The SRS conferred sugar responsiveness to a minimal promoter in an orientation-independent manner. It also converted a sugar-insensitive rice actin gene promoter into a sugar-sensitive promoter in a dose-dependent manner. Linker-scan mutation studies identified three essential motifs: the GC box, the G box, and the TATCCA element, within the SRS. Sequences containing either the GC box plus G box or the TATCCA element each mediated sugar response, however, they acted synergistically to give a high level glucose starvation-induced expression. Nuclear proteins from rice suspension cells binding to the TATCCA element in a sequence-specific and sugar-dependent manner were identified. The TATCCA element is also an important component of the gibberellin response complex of the α-amylase genes in germinating cereal grains, suggesting that the regulation of α- amylase gene expression by sugar and hormone signals may share common regulatory machinery.