Knowing the precise locations of nucleosomes in a genome is key to understanding how genes are regulated. Recent 'next generation' ChIP–chip and ChIP–Seq technologies have accelerated our understanding of the basic principles of chromatin organization. Here we discuss what high-resolution genome-wide maps of nucleosome positions have taught us about how nucleosome positioning demarcates promoter regions and transcriptional start sites, and how the composition and structure of promoter nucleosomes facilitate or inhibit transcription. A detailed picture is starting to emerge of how diverse factors, including underlying DNA sequences and chromatin remodelling complexes, influence nucleosome positioning.