Near the 5' end of most eukaryotic genes, nucleosomes form highly regular arrays that begin at canonical distances from the transcriptional start site. Determinants of this and other aspects of genomic nucleosome organization have been ascribed to statistical positioning, intrinsically DNA-encoded positioning, or some aspect of transcription initiation. Here, we provide evidence for a different explanation. Biochemical reconstitution of proper nucleosome positioning, spacing, and occupancy levels was achieved across the 5' ends of most yeast genes by adenosine triphosphate-dependent trans-acting factors. These transcription-independent activities override DNA-intrinsic positioning and maintain uniform spacing at the 5' ends of genes even at low nucleosome densities. Thus, an active, nonstatistical nucleosome packing mechanism creates chromatin organizing centers at the 5' ends of genes where important regulatory elements reside.