Our goal is to identify cis-acting elements in the regulatory region of the major seed storage protein gene in rice. A glutelin gene (pGL5-1) has been cloned by screening a rice genomic DNA library with synthetic oligonucleotides and with an amplified DNA fragment. A transient expression assay using immature rice seeds shows that its 5' flanking sequence can direct the synthesis of beta-glucuronidase (GUS) when fused upstream of the GUS coding region. Gel-retardation assays were performed to study protein-DNA interactions between putative regulatory sequences of pGL5-1 and nuclear proteins from immature rice seeds. We demonstrate that at least six protein-DNA complexes are formed between the 5' flanking sequence of pGL5-1 (-677 to -45) and nuclear protein factors. By subsequent DNase I-footprinting analyses we defined several protein-binding regions. Two of the protein-binding sequences contain the TGAGTCA motif, which is also present in the -300 element found in the 5' flanking sequences of several storage protein genes of other crop plants, and to which the transcription factors jun and GCN4 bind.