The mechanisms of biological chromatin assembly and their regulation have been studied intensively using cellular extracts, particularly those from the embryonic cells of various metazoans. Here we describe how to prepare and use a crude chromatographic fraction from budding yeast, which also supports biological chromatin assembly. In this system, nucleosomes are assembled by a replication-independent mechanism into physiologically spaced arrays that significantly protect underlying DNA from restriction endonuclease digestion. The formation of correctly spaced nucleosome arrays absolutely requires ATP and exogenous core histones of yeast or Drosophila . We have explored how cell cycle and DNA damage signals affect assembly activity in this system.