Recent genome-wide mapping of nucleosome positions revealed that well-positioned nucleosomes are pervasive across eukaryotic genomes, especially in important regulatory regions such as promoters or origins of replication. As nucleosomes impede access to DNA, their positioning is a primary mode of genome regulation. In vivo studies, especially in yeast, shed some light on factors involved in nucleosome positioning, but there is an urgent need for a complementary biochemical approach in order to confirm their direct roles, identify missing factors, and study their mechanisms. Here we describe a method that allows the genome-wide in vitro reconstitution of nucleosomes with very in vivo-like positions by a combination of salt gradient dialysis reconstitution, yeast whole cell extracts, and ATP. This system provides a starting point and positive control for the biochemical dissection of nucleosome positioning mechanisms.