Nucleosome organization is critical for gene regulation. In living cells, this organization is determined by multiple factors, including the action of chromatin remodelers, competition with site-specific DNA-binding proteins, and the DNA sequence preferences of the nucleosomes themselves. However, it has been difficult to estimate the relative importance of each of these mechanisms in vivo, because in vivo nucleosome maps reflect the combined action of all influencing factors. Here, we determine the importance of DNA sequence preferences experimentally by measuring the genome-wide occupancy of nucleosomes assembled on purified yeast genomic DNA. The resulting map, in which nucleosome occupancy is governed only by the intrinsic sequence preferences of nucleosomes, is remarkably similar to in vivo nucleosome maps generated in three different growth conditions. In vitro, nucleosome depletion is evident at many transcription factor binding sites and around gene start and end sites, suggesting that nucleosome depletion at these sites in vivo is partially encoded in the genome. We confirm these results with a micrococcal nuclease-independent experiment that measures the relative affinity of nucleosomes for ~40,000 double-stranded 150bp oligonucleotides. Using our in vitro data, we devise a computational model of nucleosome sequence preferences that is significantly correlated with in vivo nucleosome occupancy in C. elegans. Our results indicate that the intrinsic DNA sequence preferences of nucleosomes play a central role in determining the organization of nucleosomes in vivo. Overall design: Solexa/Illumina sequencing of in vivo and in vitro mononucleosomes. In vitro: 2 biological replicates YPD: 6 biological replicates (4 non-crosslinked, 2 crosslinked) YPEthanol: 4 replicates (2 non-crosslinked, 2 crosslinked) YPGalactose: 3 replicates (2 non-crosslinked, 1 crosslinked). Microarray hybridization (GPL7641) of in vitro reconstitution on synthetic sequences: Three technical replicates, pool 1 (DT sequences), pool 2 (ES sequences), pool 3 (DT and ES sequences combined). Sequencing of in vitro reconstituted nucleosomes on synthetic sequences. Input pool 1 (DT sequences, 3 technical replicates), Input pool 2 (ES sequences, 2 technical replicates), reconstituted pool 1 (3 technical replicates), reconstituted pool 2 (3 technical replicates)