The α-amylase gene is known to be regulated by the plant hormone gibberellin (GA) in cereal aleurone cells. Regulatory regions in the promoter of a rice high pl α-amylase gene, OSamy-c, were compared with that of a similar gene, RAmylA, showing the high level of sequence conservation in the proximal regions of the promoters. Expression analysis of a rice high pl α-amylase gene was carried out with a series of constructs in which 5′-deletions of the OSamy-c promoter were fused to the β-glucuronidase (GUS) gene. These constructs were introduced into rice aleurone cells by the biolistic method, which led to the identification of two functionally distinct regulatory sequences. Deletion of the negative regulatory sequence, located between -231 and -50 relative to the transcription start site, resulted in a 4-fold increase in the GUS activity. The sequence between -50 and +29 is sufficient for constitutive expression of the gene. It appears that GA-stimulated expression of the OSamy-c gene results from reversing the repression associated with the negative regulatory sequence. Gel-retardation assays identified multiple seed-specific protein factors which bind to the negative regulatory sequence between position -231 and -162. Five different proteins have been distinguished based on competitive binding studies. Three protein-binding regions have been located by footprinting analyses, one of which is located in the conserved sequence upstream of other GA-inducible genes.