Measurements of actinomycin-(3)H binding in synchronized HeLa cells reveal that the binding capacity of chromatin decreases progressively during the S phase despite a doubling of nuclear DNA content, reaches a minimal level during G(2) and mitosis, and then increases gradually throughout the subsequent G(1) interval. Since this pattern was evident in experiments with living cells, ethanol-fixed cells, and isolated nuclei, but not with purified DNA, the actinomycin binding profile may reflect changes in the degree of association between DNA and chromosomal proteins at different stages of the cell cycle.