Abstract Summary: The rapid advancement of genomic technology has revealed the enormous complexity and combinatorial nature of chromatin modifications. To facilitate interpretation of the combinatorial nature of chromatin, we have developed a novel method to integrate all chromatin datasets into distinct nucleosome types (nucleosome alphabet). We have applied this approach to Saccharomyces cerevisiae, generating a nucleosome alphabet, which forms chromatin motifs when mapped back to the genome. By applying novel chromatin alignment and global word search approaches, we have defined distinctive chromatin motifs for introns, origins of replication, tRNAs, antisense transcripts, double-strand-break hotspots and DNase hypersensitive sites, and can distinguish genes by expression level. We have also uncovered strong associations between transcription factor binding and specific types of nucleosomes. Our results demonstrate the uses and functionality of defining a chromatin alphabet and provide a unique and novel framework for exploring chromatin architecture. Contact: mjbuck@buffalo.edu Supplementary Information: Supplementary data are available at Bioinformatics online.