Gene expression and regulation in eukaryotes is controlled by orchestrated binding of regulatory proteins, including both activators and repressors, to promoters and other cis-regulatory DNA elements. An increasing number of plant genomes have been sequenced; however, a similar effort to the ENCODE project, which aimed to identify all functional elements in the human genome, has yet to be initiated in plants. Here we report genome-wide high-resolution mapping of DNase I hypersensitive (DH) sites in the model plant Arabidopsis thaliana. We identified 38,290 and 41,193 DH sites in leaf and flower tissues, respectively. The DH sites were depleted of bulk nucleosomes and were tightly associated with RNA polymerase II binding sites. Approximately 90% of the binding sites of two well-characterized MADS domain transcription factors, APETALA1 and SEPALLATA3, were covered by the DH sites. We demonstrate that protein binding footprints within a specific genomic region can be revealed using the DH site data sets in combination with known or putative protein binding motifs and gene expression data sets. Thus, genome-wide DH site mapping will be an important tool for systematic identification of all cis-regulatory DNA elements in plants.