We report a comprehensive analysis of nucleosomal and subnucleosomal organization at cis-regulatory elements (CREs) in mouse embryonic stem (ES) cells. We used chromatin fragmented by a moderate dose of micrococcal nuclease (MNase) as input for ChIP-seq with antibodies against histones and transcription factors and high-coverage deep-sequencing. Centrifugation of chromatin through sucrose gradients, followed by ChIP-seq, allowed the characterization of different subclasses of subnucleosomal particles having distribution patterns specific to enhancers, gene promoters and CTCF binding sites. We also provide datasets showing that this particular chromatin organization of CREs is conserved in the human melanoma 501Mel cell line. Overall design: We used MNase ChIP-seq to define the distribution of subnucleosomal particles, canonical nucleosomes, and transcription factors enrichment.