To investigate the mechanisms that control expression of the gene for pyruvate, orthophosphate dikinase (PPDK) in maize, the 5' flanking region of the gene was analyzed for interactions with nuclear extracts. Gel retardation assays showed that there are several sites in the promoter region which bind to protein factors. In this report we describe further study of one of these sites, designated the PPD-1 binding site. The nuclear binding factor, PPD-1, is restricted to nuclear extracts from green leaves where the PPDK gene is expressed. No binding of PPD-1 was detected in tissues such as roots or etiolated leaves where the gene is not expressed in vivo. Gel retardation assays using deletion fragments from the promoter region and synthetic oligonucleotides, as well as exonuclease III protection assays, revealed that the site of PPD-1 binding lies between positions -301 and -296. To identify the functional role of the interaction between PPD-1 and its binding site, a deletion series of the promoter region was joined to a reporter gene, beta-glucuronidase. These constructs were introduced into green leaves of maize by microprojectile bombardment. Expression of the reporter gene occurred if the PPD-1 binding site remained in the promoter region of the chimeric genes but deletion of the binding site caused a drastic reduction in expression levels. These data indicate that interaction between PPD-1 and its binding site is essential for active transcription of the PPDK gene.