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It has been reported that rice chromosome 4 has eight major heterochromatic knobs within the heterochromatic half and that this organization correlates with chromosomal-level transcriptional activity. To better understand this chromosomal organization, we created a model based on the statistical distribution of various types of gene models to divide chromosome 4 into 17 euchromatic and heterochromatic regions that correspond with the cytological staining. Fluorescence in-situ hybridization (FISH) experiments using a set of bacterial artificial chromosome (BAC) clones from chromosome 4 placed all 18 clones in the region predicted by the model. Elevated levels of H3K4 di- and tri-methylation detected by chromatin-immunoprecipitation (ChIP) on chip were correlated with euchromatic regions whereas lower levels of these two modifications were detected in heterochromatic regions. Small RNAs were more abundant in the heterochromatic regions. To validate these findings, H3K4 trimethylation, H3K9 acetylation, H4K12 acetylation, and H3K9 di- and tri-methylation of 19 individual genes were measured by ChIP–PCR. Genes in heterochromatic regions had elevated H3K9 di- and tri-methylation while genes in euchromatic regions had elevated levels of the other three modifications. We also assayed cytosine methylation of these genes using the restriction enzymes McrBC, HapII, and Msp I. This analysis indicated that cytosines of transposable elements and some genes located in heterochromatic regions were methylated while cytosines of the other genes were unmethylated. These results suggest that local transcriptional activity may reflect the organization of the corresponding part of the chromosome. They also indicate that epigenetic regulation plays an important role in correlating chromosomal organization with transcriptional activity. [ABSTRACT FROM PUBLISHER]