Regulation of gene expression is involved in the complex interaction between transcription factors and regulatory DNA cis-elements. The genomic regions associated with regulatory proteins show a status of open chromatin, which is associated with pronounced sensitivity to DNase I digestion. We generated genome-wide high resolution maps of DNase I hypersensitive (DH) sites from both seedling and callus tissues of rice (Oryza sativa). Only approximately 25% of the DH sites from both tissues were found in putative promoters. Thus, the vast majority of gene regulatory elements in the rice genome are not located in promoter regions. We found dramatic change of DH sites between seedling and callus. Genes that are differentially expressed in seedling and callus were frequently associated with DH sites in both tissues. The DNA sequences associated with the DH sites were hypomethylated. Interestingly, tissue-specific DH sites located in the promoters showed a higher level of DNA methylation than the average DNA methylation level of all DH sites located in the promoters. We also found a distinct elevation of H3K27me3 associated with intergenic DH sites. These results reveal an association between epigenetic modifications and changes of DNase I sensitivity of DH sites during development.