The question of nucleosome phasing on African Green Monkey (AGM) alpha-satellite DNA has been addressed by employing a new approach. Nucleosome cores were prepared from AGM nuclei with micrococcal nuclease, exonuclease III and nuclease S1. The core DNA population derived from alpha-satellite DNA containing chromatin was purified from total core DNA by denaturation of the DNA, reassociation to a low Cot value, and hydroxyapatite chromatography to separate the renatured satellite fraction. After end-labeling the termini of the alpha-satellite containing core DNA fragments were mapped by high resolution gel electrophoresis relative to known restriction sites along the 172 bp repeat unit of the satellite DNA. The results show that nucleosomes occupy eight strictly defined positions on the alpha-satellite DNA which could be determined with an accuracy of +/- 1 base pair. Approximately 35% of all nucleosomes are organized in one of these frames while the other seven registers contribute about 10% each.