The temporal relationships between aspects of DNA metabolism and the suppression of cell proliferation were investigated in rat glioma (strain C6) monolayer cultures exposed to 10μM dexamethasone. Cell densities (cell number per cm2), rates of DNA synthesis (dpm of [3H]thymidine incorporated per μg DNA per min), and cellular DNA (μg DNA per cm2) were measured daily in control and dexamethasone-treated cultures over a 3-day period. The percentage of cells in metaphase and the proportion of metaphases containing >2n(42) chromosomes also were determined in control and treated cultures. When log-phase C6 cultures were exposed to dexamethasone (day 0), cell densities were not significantly different from controls by day 1. Cell proliferation ceased thereafter in dexamethasone-treated cultures, whereas control cell populations continued to proliferate at log-phaserates. In contrast, cellular DNA increased exponentially in control and treated cultures over the 3-day period. On days 0 and 1, control and treated cells each contained 6 pg DNA. By day 3, the DNA content per treated cell increased to >20 pg; control cells each contained 10 pg DNA. The rates of DNA synthesis in the treated cultures did not differ significantly from controls on days 1 and 2. However, the rate in the treated cultures decreased significantly on day 3, one day after cell proliferation ceased. On day 2, the percentage of cells found in metaphase in the treated cultures was 0.32% compared to 0.64% in control cultures. By day 3, these percentages decreased to 0.20% and 0.22%, respectively. However, the proportion of metaphases containing >42 chromosomes increased 1.5-fold in the treated cultures relative to controls. These results indicate that nonproliferating dexamethasone-treated cells contain elevated amounts of DNA. Thus dexamethasone action appears to arrest the cell cycle at any point between the completion of DNA replication and mitosis.