Due to their unique structure and function, guard cells have attracted much attention at the physiological level. Very little, however, is known about the molecular events involved in the determination and maintenance of guard cell specificity. The KST1 gene encodes a K+ influx channel of guard cells in potato, and was therefore chosen as a model to study regulation of guard cell-specific gene expression. Transgenic potato plants carrying a fusion between the KST1 promoter and the E. coli uidA (beta-glucuronidase) reporter gene revealed promoter activity in guard cells and in flowers. A detailed dissection of the KST1 promoter led to the discovery of two independent small TATA box-proximal regulatory units, each of which was sufficient to direct guard cell-specific gene transcription. Both fragments contain the sequence motif, 5'-TAAAG-3', which is related to known target sites for a novel class of zinc finger transcription factors, called Dof proteins. Block mutagenesis of these Dof target sites in the context of different promoter constructs dramatically reduced guard cell promoter activity. A Dof gene, StDof1, was cloned and shown to be expressed in epidermal fragments highly enriched for guard cells. In gel retardation experiments, the StDof1 protein interacted in a sequence-specific manner with a KST1 promoter fragment containing the TAAAG motif. These results provide evidence that TAAAG elements are target sites for trans-acting Dof proteins controlling guard cell-specific gene expression. Our data will add to the design of tailor-made guard cell promoters as a further tool in molecular engineering of guard cell function and, hence, control of stomatal carbon dioxide (CO2) uptake and water loss in crop plants.