Mouse myeloid leukemia cells (M1) were induced to differentiate in vitro by treatment with dexamethasone. The incorporation of [3H]thymidine into acid-insoluble materials began to decline after 10 h treatment. This reflected the differentiation-associated decline of DNA synthesis activity since the thymidine was almost exclusively incorporated into nuclear DNA, the incorporation was completely blocked by arabinofuranosylcytosine and the differentiation-associated changes of intracellular pool size and specific activity of thymidine were negligible. Cell fractionation by discontinuous Ficoll-Urografin density gradients revealed that the DNA synthesis of the differentiated cells declined with the decreased density or ratio of nucleus size to cell size (N/C ratio). Autoradiographic analysis showed that the decrease in DNA synthesis activity was due to the accumulation of unlabeled cells, which exhibited a much lower N/C ratio than labeled cells. Dexamethasone treatment also caused specific reduction in the proportion of S phase cells. M1 cell differentiation is apparently closely coupled with the cessation of DNA synthesis.